Nucleic acid encoding a retinoblastoma binding protein (RBP-7) and polymorphic markers associated with said nucleic acid

ABSTRACT

The present invention is directed to a polynucleotide comprising open reading frames defining a coding region encoding a retinoblastoma binding protein (RBP-7) as well as regulatory regions located both at the 5′en d and the 3′ end of said coding region. The present invention also pertains to a polynucleotide carrying the natural regulation signals of the RBP-7 gene which is useful in order to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region. The invention also concerns polypeptides encoded by the coding region of the RBP-7 gene. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention also comprises genetic markers, namely biallelic markers, that are means that may be useful for the diagnosis of diseases related to an alteration in the regulation or in the coding regions of the RBP-7 gene and for the prognosis/diagnosis of an eventual treatment with therapeutic agents, especially agents acting on pathologies involving abnormal cell proliferation and/or abnormal cell differentiation

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. ProvisionalPatent Application Serial No. 60/091,315, filed Jun. 30, 1998 and U.S.Provisional Patent Application Serial No. 60/111,909, filed Dec. 10,1998, the disclosures of which are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

[0002] The present invention is directed to a polynucleotide comprisingopen reading frames defining a coding region encoding a retinoblastomabinding protein (RBP-7) as well as regulatory regions located both atthe 5′en d and the 3′ end of said coding region The present inventionalso pertains to a polynucleotide carrying the natural regulationsignals of the RBP-7 gene which is useful in order to express aheterologous nucleic acid in host cells or host organisms as well asfunctionally active regulatory polynucleotides derived from saidregulatory region. The invention also concerns polypeptides encoded bythe coding region of the RBP-7 gene. The invention also deals withantibodies directed specifically against such polypeptides that areuseful as diagnostic reagents The invention includes genetic markers,namely biallelic markers, that are means that may be useful for thediagnosis of diseases related to an alteration in the regulation or inthe coding regions of the RBP-7 gene and for the prognosis/diagnosis ofan eventual treatment with therapeutic agents, especially agents actingon pathologies involving abnormal cell proliferation and/or abnormalcell differentiation.

BACKGROUND OF THE INVENTION

[0003] Among the genetic alterations that have been shown to representdirect or indirect causative agents of proliferative diseases, such ascancers, there may be cited mutations occurring at loci harboring genesthat are called tumor suppressor genes

[0004] Tumor suppressor genes are defined as genes involved in thecontrol of abnormal cell proliferation and whose loss or inactivation isassociated with the development of malignancy Tumor suppressor genesencompass ortho-genes, emerogenes, flatogenes, and onco-suppressorgenes.

[0005] More specifically, tumor suppressor genes are genes whoseproducts inhibit cell growth. Mutant alleles in cancer cells have losttheir normal function, and act in the cell in a recessive way in thatboth copies of the gene must be inactivated in order to change the cellphenotype The tumor phenotype can be rescued by the wild-type allele, asshown by cell fusion experiments first described by Hams and colleagues(Harris H. et al., 1969). Germline mutations of tumor suppressor genesmay be transmitted and thus studied in both constitutional and tumor DNAfrom familial or sporadic cases. The current family of tumor suppressorsinclude DNA-binding transcription factors (i.e. p53, WT1), transcriptionregulators (i.e., RB, APC) and protein kinase inhibitors (i.e. p16).

[0006] The existence of tumor suppressor genes has been particularlyshown in cases of hereditary cancers These are cancer where there is aclear pattern of inheritance, usually autosomal dominant, with atendency for earlier age of onset than for sporadic tumors.

[0007] Tumor suppressor genes are detected in the form of inactivatingmutations that are tumorigenic The two best characterized genes of thisclass code for the proteins RB (Retinoblastoma protein) and p53.

[0008] Retinoblastoma is a human childhood disease, involving a tumor inthe retina. It occurs both as an inheritable trait and sporadically (bysomatic mutation). Retinoblastoma arises when both copies of the RB geneare inactivated. In the inherited form of the disease, one parentalchromosome carries an alteration in this region, usually a deletion. Asomatic event in retinal cells that causes the loss of the other copy ofthe RB gene causes a tumor. Forty percent of cases are hereditary,transmitted as an autosomal dominant trait with 90% penetrance. Of thesecases, around 10-15% are transmitted from an affected parent, theremaining arising as de novo germ-line mutations. In the sporadic formof the disease, the parental chromosomes are normal, and both RB allelesare lost by somatic events The tumor suppressor nature of RB was shownby the introduction of a single copy of RBI into tumor cell lineslacking the gene, resulting in complete or partial suppression of thetumorigenic phenotype

[0009] The RB protein has a regulatory role in cell proliferation,acting via transcription factors to prevent the transcriptionalactivation of a variety of genes, the products of which are required forthe onset of DNA synthesis, the S phase of the cell cycle

[0010] When investigating on the molecular function of RB, it has beenfound that the RB protein interacts with a variety of viral proteins,including several tumor antigens, such as SV40 T antigen, adenovirus E1Aprotein, human papillomavirus E7 These viral proteins have been shown tobind to RB, thereby inactivating it and allowing cell division to occur

[0011] Thus, an important step toward defining a mechanism underlyingtumor suppressor activity of the RB gene was the observation that thetransforming products of adenovirus (E1A protein), simian virus 40(large T antigen) and human papillomavirus (E7 protein) couldprecipitate wild-type RB protein. This, in turn, led to theidentification of a family of cellular proteins that can reversibly bindto a discrete domain on the RB protein, referred to as the T/E1A pocketby using the same specificity as the viral products The subsequentobservation that protein binding was inhibited following RB proteinphosphorylation in the late G₁ phase of the cell cycle suggested thehypothesis that the RB protein, as well as the related product p 107,may regulate the functional activity of its binding partners by acell-cycle dependent pattern of physical association. In particular, theactivity of the RB protein has been shown to be regulated through cellcycle-dependent phosphorylation by cyclin-dependent kinases.

[0012] The picture of transcription regulation is made even more complexby the finding that a number of RB related proteins (e.g. p107 and p130)also bind members of the E2F family and are therefore involved inregulatory process.

[0013] In view of the foregoing, there clearly exists a pressing need toidentify and characterize the cellular proteins that interact with theretinoblastoma protein in order to provide diagnostic and therapeutictools useful to prevent and cure cell differentiation disorders,particularly disorders in which a lack of completion of celldifferentiation, particularly in terminal cell differentiation, or inwhich an abnormal cell proliferation is detected, such as inproliferative diseases like cancer.

[0014] For the purpose of the present invention, cells with abnormalproliferation include, but are not limited to, cells characteristic ofthe following disease states: thyroid hyperplasia, psoriasis, benignprostatic hypertrophy, cancers including breast cancer, sarcomas andother neoplasms, bladder cancer, colon cancer, lung cancer, prostatecancer, various leukemias and lymphomas.

SUMMARY OF THE INVENTION

[0015] This invention is based on the discovery of a nucleic acidmolecule encoding a novel protein, more particularly a retinoblastomabinding protein (RBP-7).

[0016] The present invention pertains to nucleic acid moleculescomprising the genomic sequence of the gene encoding RBP-7. The RBP-7genomic sequence comprises regulatory sequence located upstream (5′-end)and downstream (3′-end) of the transcribed portion of said gene, theseregulatory sequences being also part of the invention

[0017] The invention also deals with the complete cDNA sequence encodingthe RBP-7 protein, as well as with the corresponding translationproduct.

[0018] Oligonucleotide probes or primers hybridizing specifically with aRBP-7 genomic or cDNA sequence are also part of the present invention,as well as DNA amplification and detection methods using said primersand probes.

[0019] A further aspect of the invention is recombinant vectorscomprising any of the nucleic acid sequences described above, and inparticular of recombinant vectors comprising a RBP-7 regulatory sequenceor a sequence encoding a RBP-7 protein, as well as of cell hosts andtransgenic non human animals comprising said nucleic acid sequences orrecombinant vectors

[0020] Finally, the invention is directed to methods for the screeningof substances or molecules that inhibit the expression of RBP-7, as wellas with methods for the screening of substances or molecules thatinteract with a RBP-7 polypeptide or that modulate the activity of aRBP-7 polypeptide

[0021] The invention also concerns biallelic markers of the RBP-7 genewhich can be useful for genetic studies, for diagnosis of diseasesrelated to an alteration in the regulation or in the coding regions ofthe RBP-7 gene and for the prognosis/diagnosis of an eventual treatmentwith therapeutic agents, especially agents acting on pathologiesinvolving abnormal cell proliferation and/or abnormal celldifferentiation

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a diagram showing a map of the RBP-7 gene.

[0023]FIG. 2 is a presentation of the RBP-7 gene structure with theamplified fragments and the biallelic markers of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

[0024] SEQ ID No. 1 contains a genomic sequence of RBP-7 comprising the5′ regulatory region (upstream untranscribed region), the exons andintrons, and the 3′ regulatory region (downstream untranscribed region)

[0025] SEQ ID No 2 contains the 5′-regulatory sequence (upstreamuntranscribed region) of RBP-7

[0026] SEQ ID No. 3 contains the 3′-regulatory sequence (upstreamuntranscribed region) of RBP-7.

[0027] SEQ ID No. 4 contains the RBP-7 cDNA sequence.

[0028] SEQ ID Nos 5 to 28 contain the exons 1 to 24 of RBP-7

[0029] SEQ ID No. 29 contains the protein sequence encoded by thenucleotide sequence of SEQ ID No 4.

[0030] SEQ ID Nos 30 to 50 contain the fragments containing apolymorphic base of a biallelic marker (first allele).

[0031] SEQ ID Nos 51 to 71 contain the fragments containing apolymorphic base of a biallelic marker (second allele).

[0032] SEQ ID Nos 72 to 101 contain the amplification primers.

[0033] SEQ ID Nos 102 to 136 contain the microsequencing primers.

[0034] SEQ ID Nos 137 and 138 contain cDNA amplification primers.

[0035] SEQ ID Nos 139 and 140 respectively contain a primer containingthe additional PU 5′ sequence and the additional RP 5′ sequencedescribed further in Example 3

[0036] In accordance with the regulations relating to Sequence Listings,the following codes have been used in the Sequence Listing to indicatethe locations of biallelic markers within the sequences and to identifyeach of the alleles present at the polymorphic base. The code “r” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is an adenine. The code “y” in thesequences indicates that one allele of the polymorphic base is athymine, while the other allele is a cytosine. The code “m” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is an cytosine. The code “k” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a thymine. The code “s” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a cytosine. The code “w” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is an thymine The nucleotide code of theoriginal allele for each biallelic marker is the following: Biallelicmarker Original allele 5-124-273 A 5-127-261 C 5-130-257 A 5-130-276 A5-131-395 A 5-135-357 A 5-136-174 T 5-140-120 T 5-143-101 C 5-143-84 G5-145-24 A 5-148-352 T 99-1437-325 A 99-1442-224 T

[0037] In some instances, the polymorphic bases of the biallelic markersalter the identity of an amino acids in the encoded polypeptide This isindicated in the accompanying Sequence Listing by use of the featureVARIANT, placement of an Xaa at the position of the polymorphic aminoacid, and definition of Xaa as the two alternative amino acids Forexample 1f one allele of a biallelic marker is the codon CAC, whichencodes histidine, while the other allele of the biallelic marker isCAA, which encodes glutamine, the Sequence Listing for the encodedpolypeptide will contain an Xaa at the location of the polymorphic aminoacid. In this instance, Xaa would be defined as being histidine orglutamine.

[0038] In other instances, Xaa may indicate an amino acid whose identityis unknown. In this instance, the feature UNSURE is used, placement ofan Xaa at the position of the unknown amino acid and definition of Xaaas being any of the 20 amino acids or being unknown.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The aim of the present invention is to provide polynucleotidesand polypeptides related to the RBP-7 gene and to a RBP-7 protein, whichis potentially involved in the regulation of the differentiation ofvarious cell types in mammals. A deregulation or an alteration of thisprotein may be involved in the generation of a pathological state in apatient. Such pathological state includes disorders caused by cellapoptosis or in contrast by an abnormal cell proliferation such as incancers.

[0040] The unphosphorylated form of the Retinoblastoma (RB) proteinspecifically binds several proteins, and these interactions occur onlyduring part of the cell cycle, prior to the S phase. The target proteinsof the RB protein include E2F transcription factors and cyclins of the Dand E types. Binding to the RB protein inhibits the ability of E2F toactivate transcription, which suggests that the RB protein may repressthe expression of genes dependent on E2F. Interaction of the RB proteinwith E2F-1, a member of the E2F transcription factors family, inhibitstranscription of genes involved in DNA synthesis and thereforesuppresses cell growth. Additionally, it has been found that thecomplexes formed between E2F and the RB protein are disrupted in thepresence of the viral oncoproteins that bind to the RB protein,suggesting a key role of the RB protein in the regulation of E2Factivity.

[0041] It has been shown that the RB protein forms two types ofcomplexes with E2F. One of these two types involves a binary complex ofthe RB protein and E2F that does not bind DNA in a gel retardationassay, and the second type of RB protein/E2F complex involves anotherfactor, RBP60, which allows the RB protein/E2F complex to bind DNA andproduce a distinct complex in a gel retardation assay. One hypothesis isthat RB protein might be regulating the DNA-binding as well as thetranscription activation function of E2F. It has also been demonstratedthat E2F can bind DNA as an oligomeric complex composed of at least twodistinct proteins.

[0042] Recent reports indicate that approximately 10 proteins have beenidentified that bind to the RB protein using the same binding surface asthe viral oncoproteins. Several of these cellular proteins, includingthe E2F transcription factor described above, comprise members of themyc oncogene family, a p46 protein (Rb-AP46), MyoD, Elf-1, proteinphosphatase type 1 catalytic subunit and several proteins designatedgenerically as “Retinoblastoma Binding Proteins” (RBBP), some of theselatter proteins being defined as E2F-like proteins.

[0043] Defeo-Jones et al. (1991) have cloned the cDNA of two members ofthe RBBP family, namely RBP-1 and RBP-2. RBP-1 and RBP-2 bindspecifically to the RB protein in vitro. RBP-2 has been shown tointeract noncovalently with RB protein via the binding of a consensusamino acid sequence of RBP-2, namely the LXCXE amino acid sequence, tothe conserved T/E1A pocket of the RB protein (Kim et al., 1994). ThisLXCXE consensus amino acid sequence is also present within theadenovirus E1A protein, the SV40 large T antigen as well as within thehuman papillomavirus E7 protein RBP-I and RBP-2 have been hypothesizedto function as transcription factors, like E2F. Helm et al (1992) havecloned a cDNA encoding another member of the RBBP family, namely RBP-3.Sakai et al. (1995) have cloned a novel RBBP protein designated asRBP-6, the locus of which has been mapped on chromosome 16 between p11.2and p12.

[0044] For the E2F family, replicating and differentiating cells needthe RB protein or RB protein family members (e.g. p107 or p130) tocounterbalance its apoptotic effect. E2F induces apoptosis whenover-expressed in cells with the wild type p53 gene, but favorsproliferation in p53 −/− cells. E2F-induced apoptosis follows entry ofthe cell into S-phase. The E2F death-promoting effect can be blocked byco-expression of p105, a RB protein family member Conversely, by geneknock-out studies, it has been demonstrated that E2F is critical for thenormal development of diverse cell types. Mice null for the E2F1 geneshow defects at a young age in the terminal differentiation of celltypes in which apoptosis play an important role, namely T-cells orepithelial cells of the testis or of other exocrine glands. Withincreasing age, these animals develop wide-spread tumors. This dataindicates that E2F plays a physiological role in normal development,probably by inducing apoptosis in a specific set of developing cells.

[0045] The retinoblastoma binding proteins of the E2F type have alsobeen described in PCT Application No. WO 65/24223, PCT Application No WO96/25494 and in U.S. Pat. No. 5,650,287, the disclosures of which areincorporated herein by reference in their entireties. Otherretinoblastoma binding proteins have been described, notably in PCTApplication No WO 94/12521, in PCT Application No WO 95/17198, in PCTApplication No. 93/23539 and in PCT Application No WO 93/06168, thedisclosures of which are incorporated herein by reference in theirentireties.

Definitions

[0046] Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

[0047] The term “RBP-7 gene”, when used herein, encompasses mRNA andcDNA sequences encoding the RBP-7 protein. In the case of a genomicsequence, the RBP-7 gene also includes native regulatory regions whichcontrol the expression of the coding sequence of the RBP-7 gene.

[0048] The term “functionally active fragment” of the RBP-7 protein isintended to designate a polypeptide carrying at least one of thestructural features of the RBP-7 protein involved in at least one of thebiological functions and/or activity of the RBP-7 protein. Particularlypreferred are peptide fragments carrying either the retinoblastomaprotein binding domain and/or the DNA binding domain of the RBP-7protein.

[0049] A “heterologous” or “exogenous” polynucleotide designates apurified or isolated nucleic acid that has been placed, by geneticengineering techniques, In the environment of unrelated nucleotidesequences, such as the final polynucleotide construct does not occurnaturally. An illustrative, but not limitatitive, embodiment of such apolynucleotide construct may be represented by a polynucleotidecomprising (1) a regulatory polynucleotide derived from the RBP-7 genesequence and (2) a polynucleotide encoding a cytokine, for exampleGM-CSF The polypeptide encoded by the heterologous polynucleotide willbe termed an heterologous polypeptide for the purpose of the presentinvention.

[0050] By a “biologically active fragment or variant” of a regulatorypolynucleotide according to the present invention is intended apolynucleotide comprising or alternatively consisting of a fragment ofsaid polynucleotide which is functional as a regulatory region forexpressing a recombinant polypeptide or a recombinant polynucleotide ina recombinant cell host

[0051] For the purpose of the invention, a nucleic acid orpolynucleotide is “functional” as a regulatory region for expressing arecombinant polypeptide or a recombinant polynucleotide if saidregulatory polynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operatively linked” to nucleotide sequences which encodethe desired polypeptide or the desired polynucleotide. An operablelinkage is a linkage in which the regulatory nucleic acid and the DNAsequence sought to be expressed are linked in such a way as to permitgene expression

[0052] As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. More precisely, twoDNA molecules (such as a polynucleotide containing a promoter region anda polynucleotide encoding a desired polypeptide or polynucleotide) aresaid to be “operably linked” if the nature of the linkage between thetwo polynucleotides does not (1) result in the introduction of aframe-shift mutation or (2) interfere with the ability of thepolynucleotide containing the promoter to direct the transcription ofthe coding polynucleotide. The promoter polynucleotide would be operablylinked to a polynucleotide encoding a desired polypeptide or a desiredpolynucleotide if the promoter is capable of effecting transcription ofthe polynucleotide of interest.

[0053] An “altered copy” of the RBP-7 gene is intended to designate aRBP-7 gene that has undergone at least one substitution, addition ordeletion of one or several nucleotides, wherein said nucleotidesubstitution, addition or deletion preferably causes a change in theamino acid sequence of the resulting translation product oralternatively causes an increase or a decrease in the expression of theRPB-7 gene

[0054] The terms “sample” or “material sample” are used herein todesignate a solid or a liquid material suspected to contain apolynucleotide or a polypeptide of the invention. A solid material maybe, for example, a tissue slice or biopsy which is searched for thepresence of a polynucleotide encoding a RBP-7 protein, either a DNA orRNA molecule or within which is searched for the presence of a native ora mutated RBP-7 protein, or alternatively the presence of a desiredprotein of interest the expression of which has been placed under thecontrol of a RBP-7 regulatory polynucleotide A liquid material may be,for example, any body fluid like serum, urine etc, or a liquid solutionresulting from the extraction of nucleic acid or protein material ofinterest from a cell suspension or from cells in a tissue slice orbiopsy. The term “biological sample” is also used and is more preciselydefined within the Section dealing with DNA extraction

[0055] As used herein, the term “purified” does not require absolutepurity; rather, it is intended as a relative definition Purification ifstarting material or natural material to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated. As an example,purification from 0.1% concentration to 10% concentration is two ordersof magnitude.

[0056] The term “isolated” requires that the material be removed fromits original environment (e g. the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition and still be isolated in thatthe vector or composition is not part of its natural environment.

[0057] Throughout the present specification, the expression “nucleotidesequence” may be employed to designate indifferently a polynucleotide oran oligonucleotide or a nucleic acid. More precisely, the expression“nucleotide sequence” encompasses the nucleic material itself and isthus not restricted to the sequence information (i.e. the succession ofletters chosen among the four base letters) that biochemicallycharacterizes a specific DNA or RNA molecule

[0058] As used interchangeably herein, the term “oligonucleotides”, and“polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of morethan one nucleotide in either single chain or duplex form. The term“nucleotide” as used herein as an adjective to describe moleculescomprising RNA, DNA, or RNA/DNA hybrid sequences of any length insingle-stranded or duplex form. The term “nucleotide” is also usedherein as a noun to refer to individual nucleotides or varieties ofnucleotides, meaning a molecule, or individual unit in a larger nucleicacid molecule, comprising a purine or pyrimidine, a ribose ordeoxyribose sugar moiety, and a phosphate group, or phosphodiesterlinkage in the case of nucleotides within an oligonucleotide orpolynucleotide. Although the term “nucleotide” is also used herein toencompass “modified nucleotides” which comprise at least onemodifications (a) an alternative linking group, (b) an analogous form ofpurine, (c) an analogous form of pyrimidine, or (d) an analogous sugar,for examples of analogous linking groups, purine, pyrimidines, andsugars see for example PCT publication No WO 95/04064 However, thepolynucleotides of the invention are preferably comprised of greaterthan 50% conventional deoxyribose nucleotides, and most preferablygreater than 90% conventional deoxyribose nucleotides. Thepolynucleotide sequences of the invention may be prepared by any knownmethod, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

[0059] The term “heterozygosity rate” is used herein to refer to theincidence of individuals in a population which are heterozygous at aparticular allele. In a biallelic system, the heterozygosity rate is onaverage equal to 2P_(a)(1−P_(a)), where P_(a) is the frequency of theleast common allele. In order to be useful in genetic studies, a geneticmarker should have an adequate level of heterozygosity to allow areasonable probability that a randomly selected person will beheterozygous.

[0060] The term “genotype” as used herein refers the identity of thealleles present in an individual or a sample. In the context of thepresent invention a genotype preferably refers to the description of thebiallelic marker alleles present in an individual or a sample. The term“genotyping” a sample or an individual for a biallelic marker consistsof determining the specific allele or the specific nucleotide carried byan individual at a biallelic marker

[0061] The term “polymorphism” as used herein refers to the occurrenceof two or more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A single nucleotide polymorphism is a single base pairchange. Typically a single nucleotide polymorphism is the replacement ofone nucleotide by another nucleotide at the polymorphic site. Deletionof a single nucleotide or insertion of a single nucleotide, also giverise to single nucleotide polymorphisms In the context of the presentinvention “single nucleotide polymorphism” preferably refers to a singlenucleotide substitution. However, the polymorphism can also involve aninsertion or a deletion of at least one nucleotide, preferably between 1and 5 nucleotides The nucleotide modification can also involve thepresence of several adjacent single base polymorphisms. This type ofnucleotide modification is usually called a ‘variable motif’. Generally,a “variable motif” involves the presence of 2 to 10 adjacent single basepolymorphisms. In some instances, series of two or more single basepolymorphisms can be interrupted by single bases which are notpolymorphic. This is also globally considered to be a “variable motif”.Typically, between different genomes or between different individuals,the polymorphic site may be occupied by two different nucleotides

[0062] The term “biallelic polymorphism” and “biallelic marker” are usedinterchangeably herein to refer to a single nucleotide polymorphismhaving two alleles at a fairly high frequency in the population. A“biallelic marker allele” refers to the nucleotide variants present at abiallelic marker site. Typically, the frequency of the less commonallele of the biallelic markers of the present invention has beenvalidated to be greater than 1%, preferably the frequency is greaterthan 10%, more preferably the frequency is at least 20% (i.e.heterozygosity rate of at least 0 32), even more preferably thefrequency is at least 30% (i e. heterozygosity rate of at least 0.42) Abiallelic marker wherein the frequency of the less common allele Is 30%or more is termed a “high quality biallelic marker”.

[0063] The location of nucleotides in a polynucleotide with respect tothe center of the polynucleotide are described herein in the followingmanner. When a polynucleotide has an odd number of nucleotides, thenucleotide at an equal distance from the 3′ and 5′ ends of thepolynucleotide is considered to be “at the center” of thepolynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter”, and so on. For polymorphisms which involve the substitution,insertion or deletion of 1 or more nucleotides, the polymorphism, alleleor biallelic marker is “at the center” of a polynucleotide if thedifference between the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 3′ end of thepolynucleotide, and the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 5′ end of thepolynucleotide is zero or one nucleotide. If this difference is 0 to 3,then the polymorphism is considered to be “within 1 nucleotide of thecenter.” If the difference is 0 to 5, the polymorphism is considered tobe “within 2 nucleotides of the center.” If the difference is 0 to 7,the polymorphism is considered to be “within 3 nucleotides of thecenter,” and so on.

[0064] As used herein the terminology “defining a biallelic marker”means that a sequence includes a polymorphic base from a biallelicmarker. The sequences defining a biallelic marker may be of any lengthconsistent with their intended use, provided that they contain apolymorphic base from a biallelic marker. The sequence is preferablybetween 1 and 500 nucleotides in length, more preferably between 5, 10,15, 20, 25, or 40 and 200 nucleotides and still more preferably between30 and 50 nucleotides in length. Each biallelic marker thereforecorresponds to two forms of a polynucleotide sequence included in agene, which, when compared with one another, present a nucleotidemodification at one position. Preferably, the sequences defining abiallelic marker include a polymorphic base selected from the groupconsisting of biallelic markers A1 to A21. In some embodiments thesequences defining a biallelic marker comprise one of the sequencesselected from the group consisting of SEQ ID Nos 30 to 71. Likewise, theterm “marker” or “biallelic marker” requires that the sequence is ofsufficient length to practically (although not necessarilyunambiguously) identify the polymorphic allele, which usually implies alength of at least 4, 5, 6, 10, 15, 20, 25, or 40 nucleotides

[0065] Variants and Fragments

[0066] 1 Polynucleotides

[0067] The invention also relates to variants and fragments of thepolynucleotides described herein, particularly of a RBP-7 genecontaining one or more biallelic markers according to the invention

[0068] Variants of polynucleotides, as the term is used herein, arepolynucleotides that differ from a reference polynucleotide A variant ofa polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical

[0069] Variants of polynucleotides according to the invention include,without being limited to, nucleotide sequences that are at least 95%identical to any of SEQ ID Nos 1-28 or the sequences complementarythereto or to any polynucleotide fragment of at least 8 consecutivenucleotides of any of SEQ ID Nos 1-28 or the sequences complementarythereto, and preferably at least 98% identical, more particularly atleast 99.5% identical, and most preferably at least 99.9% identical toany of SEQ ID Nos 1-28 or the sequences complementary thereto or to anypolynucleotide fragment of at least 8 consecutive nucleotides of any ofSEQ ID Nos 1-28 or the sequences complementary thereto.

[0070] Changes in the nucleotide of a variant may be silent, which meansthat they do not alter the amino acids encoded by the polynucleotide

[0071] However, nucleotide changes may also result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. The substitutions,deletions or additions may involve one or more nucleotides. The variantsmay be altered in coding or non-coding regions or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions.

[0072] In the context of the present invention, particularly preferredembodiments are those in which the polynucleotides encode polypeptideswhich retain substantially the same biological function or activity asthe mature RBP-7 protein

[0073] A polynucleotide fragment is a polynucleotide having a sequencethat entirely is the same as part but not all of a given nucleotidesequence, preferably the nucleotide sequence of a RBP-7 gene, andvariants thereof. The fragment can be a portion of an exon or of anintron of a RBP-7 gene it can also be a portion of the regulatorysequences of the RBP-7 gene. Preferably, such fragments comprise thepolymorphic base of at least one of the biallelic markers of SEQ ID Nos.30-71.

[0074] Such fragments may be “free-standing”, i.e. not part of or fusedto other polynucleotides, or they may be comprised within a singlelarger polynucleotide of which they form a part or region. However,several fragments may be comprised within a single larger polynucleotide

[0075] As representative examples of polynucleotide fragments of theinvention, there may be mentioned those which are from about 4, 6, 8,15, 20, 25, 40, 10 to 20, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or100 to 200 nucleotides in length Preferred are those fragments which areabout 47 nucleotides in length, such as those of SEQ ID Nos 30-71 or thesequences complementary thereto and containing at least one of thebiallelic markers of a RBP-7 gene which are described herein It will ofcourse be understood that the polynucleotides of SEQ ID Nos 30-71 or thesequences complementary thereto can be shorter or longer, although it ispreferred that they at least contain the polymorphic base of thebiallelic marker which can be located at one end of the fragment or inthe internal portion of the fragment.

[0076] 2. Polypeptides.

[0077] The invention also relates to variants, fragments, analogs andderivatives of the polypeptides described herein, including mutatedRBP-7 proteins.

[0078] The variant may be 1) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or2) one in which one or more of the amino acid residues includes asubstituent group, or 3) one in which the mutated RBP-7 is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or 4) one in which theadditional ammo acids are fused to the mutated RBP-7, such as a leaderor secretory sequence or a sequence which is employed for purificationof the mutated RBP-7 or a preprotein sequence Such variants are deemedto be within the scope of those skilled in the art.

[0079] More particularly, a variant RBP-7 polypeptide comprises aminoacid changes ranging from 1, 2, 3, 4, 5, 10 to 20 substitutions,additions or deletions of one amino acid, preferably from 1 to 10, morepreferably from 1 to 5 and most preferably from 1 to 3 substitutions,additions or deletions of one amino acid. The preferred amino acidchanges are those which have little or no influence on the biologicalactivity or the capacity of the variant RBP-7 polypeptide to berecognized by antibodies raised against a native RBP-7 protein.

[0080] As illustrative embodiments of variant RBP-7 polypeptidesencompassed by the present invention, there are the followingpolypeptides.

[0081] a polypeptide comprising a Glycine residue at the ammo acidposition 293 of the amino acid sequence of SEQ ID No. 29;

[0082] a polypeptide comprising a Glutamic acid at the amino acid inposition 963 of SEQ ID No. 29; and,

[0083] a polypeptide comprising a Methionine residue at the amino acidposition 969 of the amino acid sequence of SEQ ID No. 29.

[0084] By homologous peptide according to the present invention is meanta polypeptide containing one or several amino acid additions, deletionsand/or substitutions in the amino acid sequence of a RBP-7 polypeptide.In the case of an ammo acid substitution, one or several-consecutive ornon-consecutive-amino acids are replaced by “equivalent” amino acids.The expression “equivalent” amino acid is used herein to designate anyamino acid that may substituted for one of the amino acids belonging tothe native protein structure without decreasing the binding propertiesof the corresponding peptides to the retinoblastoma proteins (i.e. RBP,p130, p107 etc.). In other words, the “equivalent” amino acids are thosewhich allow the generation or the synthesis of a polypeptide with amodified sequence when compared to the amino acid sequence of the nativeRBP-7 protein, said modified polypeptide being able to bind to theretinoblastoma protein and/or to induce antibodies recognizing theparent polypeptide comprising, consisting essentially of, or consistingof a RBP-7 polypeptide.

[0085] These equivalent amino acids may be determined either by theirstructural homology with the initial amino acids to be replaced, by thesimilarity of their net charge, and optionally by the results of thecross-immunogenicity between the parent peptides and their modifiedcounterparts.

[0086] By an equivalent amino acid according to the present invention isalso meant the replacement of a residue in the L-form by a residue inthe D form or the replacement of a Glutamic acid (E) residue by aPyro-glutamic acid compound. The synthesis of peptides containing atleast one residue in the D-form is, for example, described by Koch (KochY., 1977, Biochem Biophys. Res Commun, Vol 74-488-491).

[0087] A specific, but not restrictive, embodiment of a modified peptidemolecule of interest according to the present invention, whichcomprises, consists essentially of, or consists of a peptide moleculewhich is resistant to proteolysis, is a peptide in which the —CONH—peptide bond is modified and replaced by a (CH₂NH) reduced bond, a(NHCO) retro inverso bond, a (CH₂—O) methylene-oxy bond, a (CH₂—S)thiomethylene bond, a (CH₂CH₂) carba bond, a (CO—CH₂) cetomethylenebond, a (CHOH—CH₂) hydroxyethylene bond), a (N—N) bound, a E-alcene bondor also a —CH═CH— bond

[0088] A polypeptide fragment is a polypeptide having a sequence thatentirely is the same as part but not all of a given polypeptidesequence, preferably a polypeptide encoded by a RBP-7 gene and variantsthereof Preferred fragments include those regions possessing antigenicproperties and which can be used to raise antibodies against the RBP-7protein

[0089] Such fragments may be “free-standing”, i.e. not part of or fusedto other polypeptides, or they may be comprised within a single largerpolypeptide of which they form a part or region However, severalfragments may be comprised within a single larger polypeptide.

[0090] As representative examples of polypeptide fragments of theinvention, there may be mentioned those which comprise at least about 5,6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 55 ammo acids ofthe RBP-7 protein. In some embodiments, the fragments contain at leastone amino acid mutation in the RBP-7 protein.

[0091] Complementary Polynucleotides

[0092] For the purpose of the present invention, a first polynucleotideis deemed to be complementary to a second polynucleotide when each basein the first polynucleotide is paired with its complementary base.Complementary bases are, generally, A and T (or A and U), or C and G.

[0093] Identity Between Nucleic Acids or Polypeptides

[0094] The terms “percentage of sequence identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e, gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Homology is evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al.,1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al.,1990, Altschul et al., 1993). In a particularly preferred embodiment,protein and nucleic acid sequence homologies are evaluated using theBasic Local Alignment Search Tool (“BLAST”) which is well known in theart (see, e g, Karlin and Altschul, 1990; Altschul et al., 1990, 1993,1997). In particular, five specific BLAST programs are used to performthe following task

[0095] (1) BLASTP and BLAST3 compare an amino acid query sequenceagainst a protein sequence database;

[0096] (2) BLASTN compares a nucleotide query sequence against anucleotide sequence database;

[0097] (3) BLASTX compares the six-frame conceptual translation productsof a query nucleotide sequence (both strands) against a protein sequencedatabase;

[0098] (4) TBLASTN compares a query protein sequence against anucleotide sequence database translated in all six reading frames (bothstrands), and

[0099] (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database.

[0100] The BLAST programs identify homologous sequences by identifyingsimilar segments, which are referred to herein as “high-scoring segmentpairs,” between a query amino or nucleic acid sequence and a testsequence which is preferably obtained from a protein or nucleic acidsequence database High-scoring segment pairs are preferably identified(i e, aligned) by means of a scoring matrix, many of which are known inthe art. Preferably, the scoring matrix used is the BLOSUM62 matrix(Gonnet et al, 1992; Henikoff and Henikoff, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978). The BLAST programs evaluate the statisticalsignificance of all high-scoring segment pairs identified, andpreferably selects those segments which satisfy a user-specifiedthreshold of significance, such as a user-specified percent homology.Preferably, the statistical significance of a high-scoring segment pairis evaluated using the statistical significance formula of Karlin (see,e.g., Karlin and Altschul, 1990) The programs listed above may be usedwith the default parameters or with modified parameters provided by theuser.

RBP-7 Gene, Corresponding cDNAs and RBP-7 Coding and RegulatorySequences

[0101] The gene encoding a RBP-7 polypeptide has been found by theinventors to be located on human chromosome 1, more precisely within the1q43 locus of said chromosome. The RBP-7 gene has a length of about 166kilobases and contains a 5′ regulatory region, 24 exons, and a 3′regulatory region. A5′-UTR region is spans the whole Exon 1 and themajor portion of the 5′ end of Exon 2. A 3′-UTR region is spans themajor portion of the 3′ end of Exon 24.

[0102] The present invention first concerns a purified or isolatednucleic acid encoding a Retinoblastoma Binding Protein named RBP-7 aswell as a nucleic acid complementary thereto and fragments and variantsthereof

[0103] In particular, the invention concerns a purified or isolatednucleic acid comprising at least 8 consecutive nucleotides of apolynucleotide selected from the group consisting of SEQ ID Nos 1 and 4as well as a nucleic acid sequence complementary thereto and fragmentsand variants thereof The length of the fragments described above canrange from at least 8, 10, 15, 20 or 30 to 200 nucleotides, preferablyfrom at least 10 to 50 nucleotides, more preferably from at least 40 to50 nucleotides. In some embodiments, the fragments may comprise morethan 200 nucleotides of SEQ ID Nos. 1 and 4 or the sequencescomplementary thereto.

[0104] The invention also pertains to a purified or isolated nucleicacid of at least 8 nucleotides in length that hybridizes under stringenthybridization conditions with a polynucleotide selected from the groupconsisting of SEQ ID Nos 1 and 4 or the sequences complementary theretoThe length of the nucleic acids described above can range from 8, 10,15, 20 or 30 to 200 nucleotides, preferably from 10 to 50 nucleotides,more preferably from 40 to 50 nucleotides Such nucleic acids may be usedas probes or primers, such as described in the corresponding section ofthe present specification.

[0105] The invention also encompasses a purified, isolated, orrecombinant polynucleotide comprising a nucleotide sequence having atleast 70, 75, 80, 85, 90, or 95% nucleotide identity with a nucleotidesequence of SEQ ID Nos 1 and 4 or a complementary sequence thereto or afragment thereof Percent identity may be determined using any of theprograms and scoring matrices described above. For example, percentidentity may be determined using BLASTN with the default parameters. Inaddition, the scoring matrix may be BLOSUM62

[0106] Particularly preferred nucleic acids of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 1 or thecomplements thereof, wherein said contiguous span comprises at least 1,2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No. 1:1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543,75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158,97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, 161453-162450. Additional preferred nucleic acids of theinvention include isolated, purified, or recombinant polynucleotidescomprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40,50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ IDNo. 4 or the complements thereof, wherein said contiguous span comprisesat least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882,4222-4276, and 5017-5579. It should be noted that nucleic acid fragmentsof any size and sequence may also be comprised by the polynucleotidesdescribed in this section.

[0107] The main structural features of the RBP-7 gene are shown inFIG. 1. The upper line shows a structural map of the polynucleotide ofSEQ ID No. 1 including the 24 exons, that are indicated by closed boxes,and the 23 introns, as well the 5′- and 3′-flanking regulatory regions.The position of the first nucleotide at 5′ end of each exon is alsoindicated, the nucleotide at position 1 being the first nucleotide atthe 5′ end of the polynucleotide of SEQ ID No 1

[0108] Generally, an intron is defined as a nucleotide sequence that ispresent both in the genomic DNA and in the unspliced mRNA molecule, andwhich is absent from the mRNA molecule which has already gone throughsplicing events.

[0109] For the purpose of the present invention and in order to make aclear and unambiguous designation of the different nucleic acidsencompassed, it has been postulated that the polynucleotides containedboth in the nucleotide sequence of SEQ ID No. 1 and in the nucleotidesequences of SEQ ID No 4 are considered as exonic sequences Conversely,the polynucleotides contained in the nucleotide sequence of SEQ ID No. 1and located between Exon 1 and Exon 24, but which are absent both fromthe nucleotide sequence of SEQ ID No. 4 are considered as intronicsequences

[0110] More precisely, the structural characteristics of the RBP-7 gene,as represented in FIG. 1 are as follows:

[0111] a) a regulatory region, located between the nucleotide atposition 1 and the nucleotide at position 273 of SEQ ID No. 1;

[0112] b) a “coding” region, located between the nucleotide at position274 and the nucleotide at position 161451 of SEQ ID No. 1, comprising 24exons and 23 introns, wherein said region defines the RBP-7 codingregion.

[0113] c) a regulatory region, beginning at the nucleotide at position161452 and ending at the nucleotide in position 162450 (the 3′-endnucleotide) of SEQ ID No 1.

[0114] The translation start site ATG is located within the second exonand the translation stop codon is located within Exon 24 of thenucleotide sequence of SEQ ID No 1.

[0115] The middle line of FIG. 1 shows the cDNA corresponding to thelongest RBP-7 mRNA including the 24 exons. Each exon is represented by aspecific box The numbers located under the exon boxes indicate thenucleotide position of the 5′ end polynucleotide of each exon, it beingunderstood that the nucleotide at position 1 is the 5 ′ end nucleotideof the cDNA, pAd denotes the four potential polyadenylation sites.

[0116] The lower line of FIG. 1 shows a map of the RBP-7 coding sequence(CDS), the start codon being located from the nucleotide in position 442to the nucleotide in position 444 of the RBP-7 cDNA of SEQ ID No. 4 andthe stop codon being located from the nucleotide in position 4378 to thenucleotide in position 4380 of the RBP-7 cDNA of SEQ ID No. 4

[0117] The 24 exons included in the RBP-7 gene are represented in FIG. 1and are described in Table A. TABLE A Begining position End positionExon SEQ ID No. in SEQ ID No 1 In SEQ ID No 1 1 5 274 665 2 6 1466 15203 7 67593 67703 4 8 71119 71184 5 9 72599 72689 6 10 75544 75623 7 1181842 81933 8 12 87902 88040 9 13 93857 93936 10 14 97159 97235 11 1598963 99117 12 16 103570 103642 13 17 105085 105179 14 18 106683 10678015 19 107799 108042 16 20 108376 108551 17 21 114336 114593 18 22 132247132331 19 23 134151 134349 20 24 145566 146774 21 25 150447 150560 22 26152960 153175 23 27 155591 155737 24 28 159702 161451

[0118] The middle line depicts the main structural features of apurified or isolated nucleic acid consisting of the longest cDNA that isobtained after reverse transcribing a mRNA generated after transcriptionof the RBP-7 gene. The longest mRNA has a nucleotide length of about 6kilobases.

[0119] As it is depicted in FIG. 1, the main characteristics of thelongest RBP-7 cDNA are the following

[0120] a) A 5′-UTR region extending from the nucleotide at position 1 tothe nucleotide at position 441 of SEQ ID No. 4,

[0121] b) An open reading frame (ORF) encoding the longest form of RBP-7protein, wherein said ORF extends from the nucleotide at position 442 tothe nucleotide at position 4380 of SEQ ID No. 4. The ATG translationstart site is located between the nucleotide at position 442 and thenucleotide at position 444 of SEQ ID No. 4. The stop codon is locatedbetween the nucleotide at position 4378 and the nucleotide at position4380 of SEQ ID No 4.

[0122] c) A 3′-UTR region extending from the nucleotide at position 4381to the nucleotide at position 6002 of SEQ ID No 4 This 3′-UTR regioncontains four potential polyadenylation sites comprising respectivelythe nucleotides between positions 4878 and 4883, 5116 and 5121, 5896 and5901 and between positions 5981 and 5986 of SEQ ID No 4

[0123]FIG. 2 is a representation of the RBP-7 gene in which the 24 exonsare shown as closed boxes.

[0124] a) In each closed box that represents a given Exon, there areindicated both a number of base pair corresponding to the non codingsequence eventually present in this Exon, and a number of amino acids.The number of amino acids is calculated as follows, starting from Exon2. Exon 2 contains two complete codons and the first base of a thirdcodon; only the two complete codons are taken into account and theadditional base is taken into account as the first base of the firstcodon of Exon 3, etc.;

[0125] b) The arrows above the Intron lines or above the Exon boxesindicate the localization of the different polymorphic markers of theinvention on the RBP-7 gene, as well as their marker names;

[0126] c) The bold letters above exons 11 and 20 indicate the effect ofthe base changes constitutive to these polymorphic markers on the aminoacid sequence of the resulting RBP-7 translation product.

[0127] The polynucleotide of SEQ ID No 4 contains, from its 5′ end toits 3′ end, the sequences resulting from the 24 exons located in Table Aon the RBP-7 genomic sequence, said exonic sequences being positioned onthe RBP-7 cDNA of SEQ ID No. 4, as detailed in Table B below TABLE B SEQID Beginning position End position Exon No. in SEQ ID No. 4 In SEQ IDNo. 4 1 5 1 392 2 6 393 447 3 7 448 558 4 8 559 624 5 9 625 715 6 10 716795 7 11 796 887 8 12 888 1026 9 13 1027 1106 10 14 1107 1183 11 15 11841338 12 16 1339 1411 13 17 1412 1507 14 18 1508 1604 15 19 1605 1848 1620 1849 2024 17 21 2025 2282 18 22 2283 2367 19 23 2368 2566 20 24 25673775 21 25 3776 3889 22 26 3890 4105 23 27 4106 4252 24 28 4253 6002

[0128] The nucleotide sequence of the RBP-7 cDNA possesses somehomologies with a cDNA encoding another human retinoblastoma bindingprotein, namely hRBP-1. This homology is randomly distributed throughoutthe whole cDNA sequences, without visible nucleic acid regions that arecharacteristic of conserved regions between cDNA sequences encodingdifferent retinobastoma binding proteins.

[0129] The majority of interrupted genes are transcribed into a RNA thatgives rise to a single type of spliced mRNA. But the RNAs of some genesfollow patterns of alternative splicing, wherein a single gene givesrise to more than one mRNA species. In some cases, the ultimate patternof expression is dictated by the primary transcript, because the use ofdifferent startpoints or termination sequences alters the splicingpattern. In other cases, a single primary transcript is spliced in morethan one way, and internal exons are substituted, added or deleted. Insome cases, the multiple products all are made in the same cell, but inothers, the process is regulated so that particular splicing patternsoccur only under particular conditions.

[0130] In the case of retinoblastoma binding proteins, alternativesplicing patterns have been observed during the processing of the RBP1pre-mRNA (Otterson et al., 1993). More precisely, alternative splicingof RBP1 clusters has been observed within a 207-nucleotide internalexon. From the four forms of mRNA detected, three of the predicted RBP1peptides share amino-terminal and carboxy-terminal domains, while afourth species encodes a distinct carboxy-terminal domain. Functionalanalysis of these peptides demonstrated that they are capable ofprecipitating retinoblastoma protein in vitro from K562 cell lysates,but cannot bind to mutant RB protein.

[0131] The inventors have found that a mRNA of about 6 kilobases andcontaining exon 1 of the RBP-7 gene at its 5′ end and exon 24 of theRBP-7 gene at its 3′ end, is produced in isolated cells from theprostate tissue, as described in Example 1.

[0132] Because the RBP-7 gene contains a large number of exons, it isexpected that the corresponding pre-mRNA is processed in a family ofmRNA molecules as a result of multiple alternative splicing events.

[0133] Additionally, individually combining each polynucleotide moleculedefining a specific exon of the RBP-7 gene with at least onepolynucleotide molecule defining another exon of the RBP-7 gene willgive rise to a family of translation products that may be assayed fortheir biological functions of interaction with retinoblastoma proteins(i e pRb, p107, p130 etc.) or of interaction with DNA sequences of thetype recognized by the transcription factors of the E2F family Suchtranslation products have a shorter size than that of the resultingprotein encoded by the longest RBP-7 mRNA and thus may be advantageouslyused in therapeutics, as compared with the longest polypeptides, due totheir weaker immunogenicity, for example

[0134] Consequently, a further aspect of the present invention is apurified or isolated nucleic acid comprising a nucleotide sequenceselected from the group consisting of SEQ ID Nos 5-28 or the sequencescomplementary thereto.

[0135] The invention also deals with a purified or isolated nucleic acidcomprising a combination of at least two polynucleotides selected fromthe group consisting of SEQ ID Nos 5-28 or the sequences complementarythereto, wherein the polynucleotides are ordered within the nucleicacid, from the 5′ end to the 3′ end of said nucleic acid, in the sameorder as in the SEQ ID No 1.

[0136] In this specific embodiment of a purified or isolated nucleicacid according to the invention, said nucleic acid preferably comprisesSEQ ID Nos 5 and 6 at its 5′ end and SEQ ID No. 28 at its 3′ end.

[0137] Regulatory Regions

[0138] As already mentioned hereinbefore, the polynucleotide of SEQ IDNo. 1 contains regulatory regions both in the non-coding 5′-flankingregion (SEQ ID No. 2) and the non-coding 3′-flanking region (SEQ ID No.3) that border the coding sequences.

[0139] The promoter activity of the regulatory region contained in SEQID No. 1 can be assessed as described below.

[0140] Genomic sequences lying upstream of the RBP-7 gene are clonedinto a suitable promoter reporter vector, such as the pSEAP-Basic,pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, or pEGFP-1 PromoterReporter vectors available from Clontech Briefly, each of these promoterreporter vectors include multiple cloning sites positioned upstream of areporter gene encoding a readily assayable protein such as secretedalkaline phosphatase, β galactosidase, or green fluorescent protein. Thesequences upstream of the RBP-7 coding region are inserted into thecloning sites upstream of the reporter gene in both orientations andintroduced into an appropriate host cell. The level of reporter proteinis assayed and compared to the level obtained from a vector which lacksan insert in the cloning site. The presence of an elevated expressionlevel in the vector containing the insert with respect to the controlvector indicates the presence of a promoter in the insert If necessary,the upstream sequences can be cloned into vectors which contain anenhancer for increasing transcription levels from weak promotersequences. A significant level of expression above that observed withthe vector lacking an insert indicates that a promoter sequence ispresent in the inserted upstream sequence.

[0141] Promoter sequences within the upstream genomic DNA may be furtherdefined by constructing nested deletions in the upstream DNA usingconventional techniques such as Exonuclease III digestion. The resultingdeletion fragments can be inserted into the promoter reporter vector todetermine whether the deletion has reduced or obliterated promoteractivity. In this way, the boundaries of the promoters may be defined.If desired, potential individual regulatory sites within the promotermay be identified using site directed mutagenesis or linker scanning toobliterate potential transcription factor binding sites within thepromoter, individually or in combination. The effects of these mutationson transcription levels may be determined by inserting the mutationsinto the cloning sites in the promoter reporter vectors.

[0142] Polynucleotides carrying the regulatory elements located both atthe 5′ end and at the 3′ end of the RBP-7 coding region may beadvantageously used to control the transcriptional and translationalactivity of an heterologous polynucleotide of interest

[0143] A 5′ regulatory polynucleotide of the invention may include the5′-untranslated region (5′-UTR) or the sequence complementary thereto,or a biologically active fragment or variant thereof The 5′-regulatorypolynucleotide harbors a CAAT box from the nucleotide in position 139 tothe nucleotide in position 147 of the nucleotide sequence of SEQ ID No.2. Additionally, the 5′-regulatory polynuceotide of the inventioncomprises a TATA box from the nucleotide in position 199 to thenucleotide in position 205 of the nucleotide sequence of SEQ ID No. 2.

[0144] A 3′ regulatory polynucleotide of the invention may include the3′-untranslated region (3′-UTR) or the sequences complementary thereto,or a biologically active fragment or variant thereof

[0145] Another aspect of the present invention is a purified and/orisolated polynucleotide located at the 5′ end of the start codon of theRBP-7 gene, wherein said polynucleotide carries expression and/orregulation signals allowing the expression of the RBP-7 gene. Thus,another part of the present invention is a purified or isolated nucleicacid comprising a nucleotide sequence of SEQ ID No. 2 and functionallyactive fragments or variants thereof The fragments may be of any lengthto facilitate the expression and/or regulation of a gene operably linkedthereto. In particular, the fragments may contain one or more bindingsites for transcription factors In some embodiments, the fragments atleast 8, 10, 15, 20 or 30 to 200 nucleotides of SEQ ID No. 2 In otherembodiments, the fragments may comprise more than 200 nucleotides of SEQID No. 2 or the sequence complementary thereto

[0146] The invention further deals with a purified and/or isolatedpolynucleotide located at the 3′ end of the stop codon of the RBP-7gene, wherein said polynucleotide carries regulation signals involved inthe expression of the RBP-7 gene Thus another part of the presentinvention is a purified or isolated nucleic acid comprising a nucleotidesequence of SEQ ID No. 3, the sequence complementary thereto, andfunctionally active fragments or variants thereof The fragments may beof any length to facilitate the expression and/or regulation of a geneoperationally linked thereto In some embodiments, the fragments maycomprise at least 8, 10, 15, 20 or 30 to 200 nucleotides of SEQ ID No. 3or the sequence complementary thereto In other embodiments, thefragments may comprise more than 200 nucleotides of SEQ ID No. 3 or thesequence complementary thereto.

[0147] Thus, the invention also pertains to a purified or isolatednucleic acid which is selected from the group consisting of:

[0148] a) a nucleic acid comprising the nucleotide sequence SEQ ID No. 2or the sequence complementary thereto;

[0149] b) a nucleic acid comprising a biologically active fragment orvariant of the nucleic acid of SEQ D No. 2 or the sequence complementarythereto.

[0150] In a specific embodiment of the above nucleic acid, said nucleicacid includes the 5′-untranslated region (5′-UTR) located between thenucleotide at position 1 to the nucleotide at position 441 of SEQ ID No.4, or the sequences complementary thereto, or a biologically activefragment or variant thereof.

[0151] Another aspect of the present invention is a purified or isolatednucleic acid which is selected from the group consisting of:

[0152] a) a nucleic acid comprising the nucleotide sequence SEQ ID No. 3or the sequence complementary thereto,

[0153] b) a nucleic acid comprising a biologically active fragment, avariant of the nucleic acid of SEQ ID No. 3 or the sequencecomplementary thereto.

[0154] In a specific embodiment of the above nucleic acid, said nucleicacid includes the 3′-untranslated region (3′-UTR) located between thenucleotide at position 4381 and the nucleotide at position 6002 of SEQID No. 4, or the sequences complementary thereto, or a biologicallyactive fragment or variant thereof

[0155] Preferred fragments of the nucleic acid of SEQ ID No. 2 or thesequence complementary thereto have a range of length from 100, 125,150, 175, 200 to 225, 250, 273 consecutive nucleotides. Preferredfragments will comprise both the CAAT box and the TATA box of thenucleotide sequence of SEQ ID No 2

[0156] Preferred fragments of the nucleic acid of SEQ ID No. 3 or thesequence complementary thereto have a length of about 600 nucleotides,more particularly of about 300 nucleotides, more preferably of about 200nucleotides and most preferably about 100 nucleotides

[0157] In order to identify the relevant biologically activepolynucleotide derivatives of SEQ ID No. 3, one may follow theprocedures described in Sambrook et al. (1989, the disclosure of whichis incorporated herein by reference) relating to the use of arecombinant vector carrying a marker gene (i.e. β galactosidase,chloramphenicol acetyl transferase, etc.) the expression of which willbe detected when placed under the control of a biologically activederivative polynucleotide of SEQ ID No. 3.

[0158] Regulatory polynucleotides of the invention may be prepared fromthe nucleotide sequence of SEQ ID No. 1 or the sequences complementarythereto by cleavage using the suitable restriction enzymes, as describedin Sambrook et al. (1989), supra.

[0159] Regulatory polynucleotides may also be prepared by digestion ofthe nucleotide sequence of SEQ ID No. 1 or the sequences complementarythereto by an exonuclease enzyme, such as Bal31 (Wabiko et al., 1986).

[0160] These regulatory polynucleotides can also be prepared by nucleicacid chemical synthesis, as described elsewhere in the specification,when oligonucleotide probes or primers synthesis is disclosed.

[0161] The regulatory polynucleotides according to the invention mayadvantageously be part of a recombinant expression vector that may beused to express a coding sequence in a desired host cell or hostorganism. The recombinant expression vectors according to the inventionare described elsewhere in the specification.

[0162] The above defined polynucleotides that carry the expressionand/or regulation signals of the RBP-7 gene may be used, for example aspart of a recombinant vector, in order to drive the expression of adesired polynucleotide, said desired polynucleotide being either (1) apolynucleotide encoding a RBP-7 protein, or a fragment or variantthereof, or (2) an “heterologous” polynucleotide, such as apolynucleotide encoding a desired “heterologous” polypeptide or adesired RNA in a recombinant cell host

[0163] The invention also encompasses a polynucleotide comprising,consisting essentially of, or consisting of:

[0164] a) a nucleic acid comprising a regulatory polynucleotide of SEQID No 2, or the sequence complementary thereto, or a biologically activefragment or variant thereof,

[0165] b) a polynucleotide encoding a desired polypeptide or nucleicacid

[0166] c) Optionally, a nucleic acid comprising a regulatorypolynucleotide of SEQ ID No 3, or the sequence complementary thereto, ora biologically active fragment or variant thereof

[0167] In a preferred embodiment, a polynucleotide such as disclosedabove comprises the nucleic acid of SEQ ID No 2, or the sequencescomplementary thereto, or a fragment, a variant or a biologically activederivative thereof which is located at the 5′ en d of the polynucleotideencoding the desired polypeptide or polynucleotide.

[0168] In another embodiment, a polynucleotide such as that abovedescribed comprises the nucleic acid of SEQ ID No. 3, or the sequencecomplementary thereto, or a fragment, a variant or a biologically activederivative thereof which is located at the 3′ end of the polynucleotideencoding the desired polypeptide or nucleic acid. A preferred desirednucleic acid comprises of a ribonucleic acid useful as antisensemolecule.

[0169] The desired polypeptide encoded by the above described nucleicacid may be of various nature or origin, encompassing proteins ofprokaryotic or eukaryotic origin. Among the polypeptides which may beexpressed under the control of a RBP-7 regulatory region are bacterial,fungal or viral antigens. Are also encompassed eukaryotic proteins suchas intracellular proteins, such as “house keeping” proteins,membrane-bound proteins, such as receptors, and secreted proteins suchas the numerous endogenous mediators including cytokines.

[0170] The desired nucleic acid encoded by the above describedpolynucleotide, usually a RNA molecule, may be complementary to a RBP-7coding sequence and thus useful as an antisense polynucleotide.

[0171] Such a polynucleotide may be included in a recombinant expressionvector in order to express a desired polypeptide or a desiredpolynucleotide in host cell or in a host organism. Suitable recombinantvectors that contain a polynucleotide such as described hereinbefore aredisclosed elsewhere in the specification.

[0172] Coding Regions

[0173] As depicted in FIG. 1, the RBP-7 open reading frame is containedin the longest RBP-7 which mRNA has a nucleotide length of about 4kilobases.

[0174] More precisely, the effective RBP-7 coding sequence (CDS) isbetween the nucleotide at position 442 and the nucleotide at position4377 of SEQ ID No 4

[0175] The invention further provides a purified or isolated nucleicacid comprising a polynucleotide selected from the group consisting of apolynucleotide comprising a nucleic acid sequence located between thenucleotide at position 442 and the nucleotide at position 4377 of SEQ IDNo 4, or the sequence complementary thereto, or a variant or fragmentthereof or a sequence complementary thereto.

[0176] A further object of the present invention comprisespolynucleotide fragments of the RBP-7 gene that are useful for thedetection of the presence of an unaltered or an altered copy of theRBP-7 gene within the genome of a host organism and also for thedetection and/or quantification of the expression of the RBP-7 gene insaid host organism.

[0177] Thus, another object of the present invention is a purified orisolated nucleic acid encoding a variant or a mutated RBP-7 protein

[0178] A first preferred embodiment of a copy of the RBP-7 genecomprises an allele in which a single base substitution in the codonencoding the Aspartic acid (D) residue in amino acid position 293 of theRBP-7 protein of SEQ ID No. 29 leads to the amino acid replacement for aGlycine (G) residue.

[0179] A second preferred embodiment of a copy of the RBP-7 genecomprises an allele in which a single base substitution in the codonencoding the Glycine (G) residue in amino acid position 963 of the RBP-7protein of SEQ ID No. 29 leads to the amino acid replacement for aGlutamic acid (E) residue.

[0180] A third preferred embodiment of a copy of the RBP-7 genecomprises an allele in which a single base substitution in the codonencoding the Leucine (L) residue in amino acid position 969 of the RBP-7protein of SEQ ID No. 29 leads to the amino acid replacement for aMethionine (M) residue.

[0181] Thus, another object of the present invention is a purified orisolated nucleic acid encoding a mutated RBP-7 protein.

[0182] The above disclosed polynucleotide that contains only codingsequences derived from the RBP-7 ORF may be expressed in a desired hostcell or a desired host organism, when said polynucleotide is placedunder the control of suitable expression signals Such a polynucleotide,when placed under the suitable expression signals, may be inserted in avector for its expression.

Oligonucleotide Probes and Primers

[0183] Polynucleotides derived from the RBP-7 gene described above areuseful in order to detect the presence of at least a copy of anucleotide sequence of SEQ ID No. 1, or a fragment or a variant thereofin a test sample

[0184] The present invention concerns a purified or isolated nucleicacid comprising at least 8 consecutive nucleotides of the nucleotidesequence SEQ ID No. 1 or a sequence complementary thereto or variantsthereof In another embodiment, the present invention relates to nucleicacids comprising at least 8, 10, 15, 20 or 30 to 200 nucleotides,preferably from at least 10 to 50 nucleotides, more preferably from atleast 40 to 50 nucleotides of SEQ ID No 1 or the sequence complementarythereto. In some embodiments, the nucleic acids may comprise more than200 nucleotides of SEQ ID No. 1 or the sequence complementary thereto.

[0185] Particularly preferred probes and primers of the inventioninclude isolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 1 or thecomplements thereof, wherein said contiguous span comprises at least 1,2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No11-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543,75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158,97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, 161453-162450.

[0186] The invention also relates to an oligonucleotide of at least atleast 8 nucleotides in length that hybridizes under stringenthybridization conditions with a nucleic acid selected from the groupconsisting of the nucleotide sequences 1-481, 666-1465, 1521-67592,67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019,83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188,103745-104303, 104654-105084, 105180-106682, 106781-107798,107897-108392, 108552-114335, 114418-114491, 114594-132246,132332-134150, 134350-145565, 145842-146332, 146775-150446,150542-152959, 153176-155590, 155738-159701, 160466-161028,161453-162450 of SEQ ID No. 1 or a variant thereof or a sequencecomplementary thereto. In some embodiments, the invention relates tosequences comprising at least 8, 10, 15, 20 or 30 to 200 nucleotides,preferably from at least 10 to 50 nucleotides, more preferably from 40to 50 nucleotides of SEQ ID No. 1 or the sequence complementary theretoor variants thereof. In some embodiments, the invention relates tosequences comprising more than 200 nucleotides of SEQ ID No. 1 or thesequence complementary thereto

[0187] For the purpose of defining such a hybridizing nucleic acidaccording to the invention, the stringent hybridization conditions arethe following:

[0188] the hybridization step is realized at 65° C. in the presence of6×SSC buffer, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml of salmonsperm DNA.

[0189] The hybridization step is followed by four washing steps:

[0190] two 5 min washings, preferably at 65C in a 2×SSC and 0.1%SDSbuffer;

[0191] one 30 mm washing, preferably at 65° C. in a 2×SSC and 0 1% SDSbuffer,

[0192] one 10 mm washing, preferably at 65° C. in a 0.1×SSC and 0.1%SDSbuffer,

[0193] the above hybridization conditions are suitable for a nucleicacid molecule of about 20 nucleotides in length. There is no need to saythat the hybridization conditions described above can readily be adaptedaccording to the length of the desired nucleic acid, followingtechniques well known to the one skilled in the art. The hybridizationconditions may for example be adapted according to the teachingsdisclosed in the book of Hames and Higgins (1985), the disclosure ofwhich is incorporated herein by reference.

[0194] Another aspect of the invention is a purified or isolated nucleicacid comprising at least 8 consecutive nucleotides of the nucleotidesequence SEQ ID No 4 or the sequence complementary thereto or variantsthereof. In another embodiment, the nucleic acid comprises from at least8, 10, 15, 20 or 30 to 200 nucleotides, preferably from at least 10 to50 nucleotides, more preferably from at least 40 to 50 nucleotides ofSEQ ID No. 4 or the sequence complementary thereto or variants thereof.In some embodiments, the fragments may comprise more than 200nucleotides of SEQ ID No. 4 or the sequence complementary thereto orvariants thereof

[0195] Additional preferred probes and primers of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 4 or thecomplements thereof, wherein said contiguous span comprises at least 1,2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No. 4:1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276,and 5017-5579.

[0196] Alternatively, the invention also relates to an oligonucleotideof at least 8 nucleotides in length that hybridizes under the stringenthybridization conditions previously defined with a nucleic acid selectedfrom the group consisting of the nucleotide sequences 1-208, 1307-1350,1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579 ofSEQ ID No. 1 or a variant thereof or a sequence complementary thereto.

[0197] A nucleic probe or primer according to the invention comprises atleast 8 consecutive nucleotides of a polynucleotide of SEQ ID Nos 1 or 4or the sequences complementary thereto, preferably from 8 to 200consecutive nucleotides, more particularly from 10, 15, 20 or 30 to 100consecutive nucleotides, more preferably from 10 to 50 nucleotides, andmost preferably from 40 to 50 consecutive nucleotides of apolynucleotide of SEQ ID Nos 1 or 4 or the sequences complementarythereto.

[0198] In a first preferred embodiment, the probe or primer is suspendedin a suitable buffer for performing a hybridization or an amplificationreaction

[0199] In a second embodiment, the oligonucleotide probe, which may beimmobilized on a support, is capable of hybridizing with a RBP-7 gene,preferably with a region of the RBP-7 gene which comprises a biallelicmarker of the present invention. The techniques for immobilizing anucleotide primer or probe on a solid support are well-known to theskilled artisan and include, but are not limited to, the immobilizationtechniques described in the present application

[0200] In a third embodiment, the primer is complementary to anynucleotide sequence of the RBP-7 gene and can be used to amplify aregion of the RBP-7 gene contained in the nucleic acid sample to betested which includes a polymorphic base of at least one biallelicmarker. Preferably, the amplified region includes a polymorphic base ofat least one biallelic marker selected from the group consisting of SEQID Nos 30-71 or the sequences complementary thereto. In someembodiments, the primer comprises one of the sequences of SEQ ID Nos72-101 and 102-136.

[0201] When using a polynucleotide probe or primer in a detection methodof the invention, the DNA or RNA contained in the sample to be assayedmay be subjected to a first extraction step well known to the oneskilled in the art, in order to make the DNA or RNA material containedin the initial sample available to a hybridization reaction, prior tothe hybridization step itself

[0202] The nucleic acid probes and primers of the invention are alsoused to detect and/or amplify a portion of the RBP-7 gene within which apolymorphism or a mutation causes a change either in the expressionlevel of the RBP-7 gene or a change in the amino acid sequence of theRBP-7 gene translation product.

[0203] The invention further concerns detection or amplification kitscontaining a pair of oligonucleotide primers or an oligonucleotide probeaccording to the invention. The kits of the present invention can alsocomprise optional elements including appropriate amplification reagentssuch as DNA polymerases when the kit comprises primers, or reagentsuseful in hybridization between a labeled hybridization probe and aRBP-7 gene containing at least one biallelic marker. In one embodiment,the biallelic marker comp rises one of the sequences of SEQ ID Nos 30-71or the sequences complementary thereto

[0204] In one embodiment the invention encompasses isolated, purified,and recombinant polynucleotides comprising, consisting of, or consistingessentially of a contiguous span of 8 to 50 nucleotides of any one ofSEQ ID Nos 1 and 4 and the complement thereof, wherein said spanincludes a biallelic marker of RBP-7 in said sequence; optionally,wherein said biallelic marker of RBP-7 is selected from the groupconsisting of A1 to A21, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith; optionally,wherein said contiguous span is 18 to 47 nucleotides in length and saidbiallelic marker is within 4 nucleotides of the center of saidpolynucleotide, optionally, wherein said polynucleotide consists of orcomprises said contiguous span and said contiguous span is 25nucleotides in length and said biallelic marker is at the center of saidpolynucleotide; optionally, wherein the 3′ end of said contiguous spanis present at the 3′ end of said polynucleotide, and optionally, whereinthe 3′ end of said contiguous span is located at the 3′ end of saidpolynucleotide and said biallelic marker is present at the 3′ end ofsaid polynucleotide. In a preferred embodiment, said probes comprises,consists of, or consists essentially of a sequence selected from thesequences SEQ ID Nos 30-71 and the complementary sequences thereto.

[0205] In another embodiment the invention encompasses isolated,purified and recombinant polynucleotides comprising, consisting of, orconsisting essentially of a contiguous span of 8 to 50 nucleotides ofSEQ ID Nos 1 and 4 or the complements thereof, wherein the 3′ end ofsaid contiguous span is located at the 3′ end of said polynucleotide,and wherein the 3′ end of said polynucleotide is located within 20nucleotides upstream of a biallelic marker of RBP-7 in said sequence;optionally, wherein said biallelic marker of RBP-7 is selected from thegroup consisting of A1 to A21, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, wherein the 3′ end of said polynucleotide is located 1nucleotide upstream of said biallelic marker of RBP-7 in said sequence;and optionally, wherein said polynucleotide comprises, consists of, orconsists essentially of a sequence selected from the sequences SEQ IDNos 102-136

[0206] In a further embodiment, the invention encompasses isolated,purified, or recombinant polynucleotides comprising, consisting of, orconsisting essentially of a sequence selected from the sequences SEQ IDNos 72-101.

[0207] In an additional embodiment, the invention encompassespolynucleotides for use m hybridization assays, sequencing assays, andenzyme-based mismatch detection assays for determining the identity ofthe nucleotide at a biallelic marker of RBP-7 in SEQ ID Nos 1 and 4, orthe complements thereof, as well as polynucleotides for use inamplifying segments of nucleotides comprising a biallelic marker ofRBP-7 in SEQ ID Nos 1 and 4, or the complements thereof; optionally,wherein said biallelic marker of RBP-7 is selected from the groupconsisting of A1 to A21, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith

[0208] The formation of stable hybrids depends on the meltingtemperature (Tm) of the DNA The Tm depends on the length of the primeror probe, the ionic strength of the solution and the G+C content Thehigher the G+C content of the primer or probe, the higher is the meltingtemperature because G:C pairs are held by three H bonds whereas A.Tpairs have only two. The GC content in the probes and primers of theinvention usually ranges between 10 and 75%, preferably between 35 and60%, and more preferably between 40 and 55%

[0209] The length of these probes and probes can range from 8, 10, 15,20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferablyfrom 15 to 30 nucleotides. Shorter probes and primers tend to lackspecificity for a target nucleic acid sequence and generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. Longer probes and primers are expensive to produce and cansometimes self-hybridize to form hairpin structures. The appropriatelength for primers and probes under a particular set of assay conditionsmay be empirically determined by one of skill in the art

[0210] The primers and probes can be prepared by any suitable method,including, for example, cloning and restriction of appropriate sequencesand direct chemical synthesis by a method such as the phosphodiestermethod of Narang et al. (1979), the phosphodiester method of Brown etal. (1979), the diethylphosphoramidite method of Beaucage et al. (1981)and the solid support method described in EP 0 707 592, the disclosuresof which are incorporated herein by reference in their entireties.

[0211] Any of the polynucleotides of the present invention can belabeled, if desired, by incorporating a label detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include radioactive substances (³²P,³⁵S, ³H, ¹²⁵I), fluorescent dyes (5-bromodesoxyuridin, fluorescein,acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotidesare labeled at their 3′ and 5′ ends. Examples of non-radioactivelabeling of nucleic acid fragments are described in the French PatentNo. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988)Advantageously, the probes according to the present invention may havestructural characteristics such that they allow the signalamplification, such structural characteristics being, for example,branched DNA probes as those described by Urdea et al. in 1991 or in theEuropean Patent No. EP-0225,807, the disclosure of which is incorporatedherein by reference in its entirety (Chiron).

[0212] A label can also be used to capture the primer, so as tofacilitate the immobilization of either the primer or a primer extensionproduct, such as amplified DNA, on a solid support. A capture label isattached to the primers or probes and can be a specific binding memberwhich forms a binding pair with the solid's phase reagent's specificbinding member (e g. biotin and streptavidin). Therefore depending uponthe type of label carried by a polynucleotide or a probe, it may beemployed to capture or to detect the target DNA Further, it will beunderstood that the polynucleotides, primers or probes provided herein,may, themselves, serve as the capture label. For example, in the casewhere a solid phase reagent's binding member is a nucleic acid sequence,it may be selected such that it binds a complementary portion of aprimer or probe to thereby immobilize the primer or probe to the solidphase In cases where a polynucleotide probe itself serves as the bindingmember, those skilled in the art will recognize that the probe willcontain a sequence or “tall” that is not complementary to the target. Inthe case where a polynucleotide primer itself serves as the capturelabel, at least a portion of the primer will be free to hybridize with anucleic acid on a solid phase DNA Labeling techniques are well known tothe skilled technician

[0213] The probes of the present invention are useful for a number ofpurposes. They can be notably used in Southern hybridization to genomicDNA The probes can also be used to detect PCR amplification productsThey may also be used to detect mismatches in the RBP-7 gene or mRNAusing other techniques.

[0214] Any of the polynucleotides, primers and probes of the presentinvention can be conveniently immobilized on a solid support. Solidsupports are known to those skilled in the art and include the walls ofwells of a reaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, duracytes andothers. The solid support is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and duracytes are all suitable examples. Suitable methods forimmobilizing nucleic acids on solid phases include ionic, hydrophobic,covalent interactions and the like. A solid support, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid support can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid support and which has the ability to immobilize thecapture reagent through a specific binding reaction. The receptormolecule enables the indirect binding of the capture reagent to a solidsupport material before the performance of the assay or during theperformance of the assay. The solid phase thus can be a plastic,derivatized plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microtiter well, sheet, bead, microparticle,chip, sheep (or other suitable animal's) red blood cells, duracytes® andother configurations known to those of ordinary skill in the art. Thepolynucleotides of the invention can be attached to or immobilized on asolid support individually or in groups of at least 2, 5, 8, 10, 12, 15,20, or 25 distinct polynucleotides of the inventions to a single solidsupport In addition, polynucleotides other than those of the inventionmay attached to the same solid support as one or more polynucleotides ofthe invention

[0215] Consequently, the invention also deals with a method fordetecting the presence of a nucleic acid comprising a nucleotidesequence selected from a group consisting of SEQ ID Nos 1, 4, a fragmentor a variant thereof or the complementary sequence thereto in a sample,said method comprising the following steps of:

[0216] a) bringing into contact a nucleic acid probe or a plurality ofnucleic acid probes as described above and the sample to be assayed

[0217] b) detecting the hybrid complex formed between the probe and anucleic acid in the sample.

[0218] In a first preferred embodiment of this detection method, saidnucleic acid probe or the plurality of nucleic acid probes are labeledwith a detectable molecule.

[0219] In a second preferred embodiment of said method, said nucleicacid probe or the plurality of nucleic acid probes has been immobilizedon a substrate.

[0220] The invention further concerns a kit for detecting the presenceof a nucleic acid comprising a nucleotide sequence selected from a groupconsisting of SEQ ID Nos 1, 4, a fragment or a variant thereof or thecomplementary sequence thereto in a sample, said kit comprising:

[0221] a) a nucleic acid probe or a plurality of nucleic acid probes asdescribed above;

[0222] b) optionally, the reagents necessary for performing thehybridization reaction.

[0223] In a first preferred embodiment of the detection kit, the nucleicacid probe or the plurality of nucleic acid probes are labeled with adetectable molecule.

[0224] In a second preferred embodiment of the detection kit, thenucleic acid probe or the plurality of nucleic acid probes has beenimmobilized on a substrate.

[0225] Oligonucleotide Arrays

[0226] A substrate comprising a plurality of oligonucleotide primers orprobes of the invention may be used either for detecting or amplifyingtargeted sequences in the RBP-7 gene and may also be used for detectingmutations in the coding or in the non-coding sequences of the RBP-7gene.

[0227] Any polynucleotide provided herein may be attached in overlappingareas or at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assay procedureAddressable polynucleotide arrays typically comprise a plurality ofdifferent oligonucleotide probes that are coupled to a surface of asubstrate in different known locations. The knowledge of the preciselocation of each polynucleotides location makes these “addressable”arrays particularly useful in hybridization assays Any addressable arraytechnology known in the art can be employed with the polynucleotides ofthe invention. One particular embodiment of these polynucleotide arraysis known as the Genechips™, and has been generally described in U.S.Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, thedisclosures of which are incorporated herein by reference in theirentireties. These arrays may generally be produced using mechanicalsynthesis methods or light directed synthesis methods which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis (Fodor et al., Science, 251.767-777, 1991).The immobilization of arrays of oligonucleotides on solid supports hasbeen rendered possible by the development of a technology generallyidentified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™)in which, typically, probes are immobilized in a high density array on asolid surface of a chip. Examples of VLSIPS™ technologies are providedin U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO90/15070, WO 92/10092 and WO 95/11995, the disclosures of which areincorporated herein by reference in their entireties, which describemethods for forming oligonucleotide arrays through techniques such aslight-directed synthesis techniques. In designing strategies aimed atproviding arrays of nucleotides immobilized on solid supports, furtherpresentation strategies were developed to order and display theoligonucleotide arrays on the chips in an attempt to maximizehybridization patterns and sequence information. Examples of suchpresentation strategies are disclosed in PCT Publications WO 94/12305,WO 94/11530, WO 97/29212 and WO 97/31256, the disclosures of which areincorporated herein by reference in their entireties.

[0228] In another embodiment of the oligonucleotide arrays of theinvention, an oligonucleotide probe matrix may advantageously be used todetect mutations occurring in the RBP-7 gene and in its regulatoryregion. For this particular purpose, probes are specifically designed tohave a nucleotide sequence allowing their hybridization to the genesthat carry known mutations (either by deletion, insertion ofsubstitution of one or several nucleotides) By known mutations is meantmutations on the RBP-7 gene that have been identified according, forexample to the technique used by Huang et al. (1996) or Samson et al.(1996).

[0229] Another technique that is used to detect mutations in the RBP-7gene is the use of a high-density DNA array. Each oligonucleotide probeconstituting a unit element of the high density DNA array is designed tomatch a specific subsequence of the RBP-7 genomic DNA or cDNA Thus, anarray comprising, consisting essentially of, or consisting ofoligonucleotides complementary to subsequences of the target genesequence is used to determine the identity of the target sequence withthe wild gene sequence, measure its amount, and detect differencesbetween the target sequence and the reference wild gene sequence of theRBP-7 gene. One such design, termed 4L tiled array, uses a set of fourprobes (A, C, G, T), preferably 15-nucleotide oligomers. In each set offour probes, the perfect complement will hybridize more strongly thanmismatched probes. Consequently, a nucleic acid target of length L isscanned for mutations with a tiled array containing 4L probes, the wholeprobe set containing all the possible mutations in the known wildreference sequence. The hybridization signals of the 15-mer probe settiled array are perturbed by a single base change in the targetsequence. As a consequence, there is a characteristic loss of signal ora “footpnnt” for the probes flanking a mutation position. This techniquewas described by Chee et al. m 1996, which is herein incorporated byreference.

[0230] Consequently, the invention concerns an array of nucleic acidcomprising at least one polynucleotide described above as probes andprimers. Preferably, the invention concerns an array of nucleic acidcomprising at least two polynucleotides described above as probes andprimers

Amplification of the RBP-7 Gene

[0231] 1. DNA Extraction

[0232] As for the source of the genomic DNA to be subjected to analysis,any test sample can be foreseen without any particular limitation. Thesetest samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants, fixed tissue specimens including tumor andnon-tumor tissue and lymph node tissues; bone marrow aspirates and fixedcell specimens. The preferred source of genomic DNA used in the contextof the present invention is from peripheral venous blood of each donor.

[0233] The techniques of DNA extraction are well-known to the skilledtechnician. Such techniques are described notably by Lin et al. (1998)and by Mackey et al. (1998).

[0234] 2. DNA Amplification

[0235] DNA amplification techniques are well-known to those skilled inthe art. Amplification techniques that can be used in the context of thepresent invention include, but are not limited to, the ligase chainreaction (LCR) described in EP-A-320 308, WO 9320227 and EP-A-439 182,the disclosures of which are incorporated herein by reference, thepolymerase chain reaction (PCR, RT-PCR) and techniques such as thenucleic acid sequence based amplification (NASBA) described in GuatelliJ C, et al. (1990) and in Compton J (1991), Q-beta amplification asdescribed in European Patent Application No. 4544610, stranddisplacement amplification as described in Walker et al. (1996) and EP A684 315 and, target mediated amplification as described in PCTPublication WO 9322461, the disclosure of which is incorporated hereinby reference

[0236] LCR and Gap LCR are exponential amplification techniques, bothdepend on DNA ligase to join adjacent primers annealed to a DNAmolecule. In Ligase Chain Reaction (LCR), probe pairs are used whichinclude two primary (first and second) and two secondary (third andfourth) probes, all of which are employed in molar excess to target. Thefirst probe hybridizes to a first segment of the target strand and thesecond probe hybridizes to a second segment of the target strand, thefirst and second segments being contiguous so that the primary probesabut one another in 5′ phosphate-3′ hydroxyl relationship, and so that aligase can covalently fuse or ligate the two probes into a fusedproduct. In addition, a third (secondary) probe can hybridize to aportion of the first probe and a fourth (secondary) probe can hybridizeto a portion of the second probe in a similar abutting fashion. Ofcourse, if the target is initially double stranded, the secondary probesalso will hybridize to the target complement in the first instance. Oncethe ligated strand of primary probes is separated from the targetstrand, it will hybridize with the third and fourth probes, which can beligated to form a complementary, secondary ligated product. It isimportant to realize that the ligated products are functionallyequivalent to either the target or its complement By repeated cycles ofhybridization and ligation, amplification of the target sequence isachieved. A method for multiplex LCR has also been described (WO9320227) Gap LCR (GLCR) is a version of LCR where the probes are notadjacent but are separated by 2 to 3 bases.

[0237] For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR), or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR(RT-AGLCR) as described by Marshall et al. (1994). AGLCR is amodification of GLCR that allows the amplification of RNA.

[0238] The PCR technology is the preferred amplification technique usedin the present invention A variety of PCR techniques are familiar tothose skilled in the art. For a review of PCR technology, see White(1997) and the publication entitled “PCR Methods and Applications”(1991, Cold Spring Harbor Laboratory Press) In each of these PCRprocedures, PCR primers on either side of the nucleic acid sequences tobe amplified are added to a suitably prepared nucleic acid sample alongwith dNTPs and a thermostable polymerase such as Taq polymerase, Pfupolymerase, or Vent polymerase. The nucleic acid in the sample isdenatured and the PCR primers are specifically hybridized tocomplementary nucleic acid sequences in the sample. The hybridizedprimers are extended. Thereafter, another cycle of denaturation,hybridization, and extension is initiated The cycles are repeatedmultiple times to produce an amplified fragment containing the nucleicacid sequence between the primer sites. PCR has further been describedin several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and4,965,188 Each of these publications is incorporated by reference.

[0239] One of the aspects of the present invention is a method for theamplification of the human RBP-7 gene, particularly of the genomicsequences of SEQ D) No. 1 or of the cDNA sequence of SEQ ID No. 4, or afragment or a variant thereof in a test sample, preferably using the PCRtechnology. The method comprises the steps of contacting a test samplesuspected of containing the target RBP-7 encoding sequence or portionthereof with amplification reaction reagents comprising a pair ofamplification primers, and eventually in some instances a detectionprobe that can hybridize with an internal region of amplicon sequencesto confirm that the desired amplification reaction has taken place

[0240] Thus, the present invention also relates to a method for theamplification of a human RBP-7 gene sequence, particularly of a portionof the genomic sequences of SEQ ID No. 1 or of the cDNA sequence of SEQID No. 4, or a variant thereof in a test sample, said method comprisingthe steps of:

[0241] a) contacting a test sample suspected of containing the targetedRBP-7 gene sequence comprised in a nucleotide sequence selected from agroup consisting of SEQ ID Nos 1 and 4, or fragments or variants thereofwith amplification reaction reagents comprising a pair of amplificationprimers as described above and located on either side of thepolynucleotide region to be amplified, and

[0242] b) optionally detecting the amplification products.

[0243] In a preferred embodiment of the above amplification method, theamplification product is detected by hybridization with a labeled probehaving a sequence which is complementary to the amplified region.

[0244] The primers are more particularly characterized in that they havesufficient complementarity with any sequence of a strand of the genomicsequence close to the region to be amplified, for example with anon-coding sequence adjacent to exons to amplify.

[0245] In a particular embodiment of the invention, the primers areselected form the group consisting of the nucleotide sequences detailedin Table C below. TABLE C Forward Position range of ReverseComplementary position Primer amplification primer Primer range ofamplification Name in SEQ ID No. 1 Name primer in SEQ ID No. 1 P1313-330 P26 732-751 P2 1282-1299 P27 1682-1699 P3 67531-67549 P2867810-67830 P4 70927-70945 P29 71257-71276 P5 71613-71631 P3072043-72060 P6 75390-75409 P31 75795-75814 P7 77544-77563 P3277926-77943 P8 81708-81726 P33 82108-82127 P9 105046-105065 P34105326-105345 P10 104751-104770 P35 105297-105316 P11 107691-107710 P36108091-108110 P12 114296-114315 P37 114698-114716 P13 114327-114345 P38114735-114753 P14 132101-132118 P39 132504-132521 P15 145522-145541 P40145923-145942 P16 145866-145884 P41 146266-146285 P17 145956-145976 P42146399-146418 P18 146529-146547 P43 146955-146972 P19 152763-152780 P44153164-153182 P20 155404-155422 P45 155706-155726 P21 160043-160060 P46160445-160462 P22 160361-160378 P47 160770-160788 P23 160742-160759 P48161147-161165 P24 161127-161144 P49 161530-161547 P25 161217-161235 P50161617-161636

[0246] The invention also concerns a kit for the amplification of ahuman RBP-7 gene sequence, particularly of a portion of the genomicsequences of SEQ ID No. 1 or of the cDNA sequence of SEQ ID No. 4, or avariant thereof in a test sample, wherein said kit comprises:

[0247] a) A pair of oligonucleotide primers located on either side ofthe RBP-7 region to be amplified;

[0248] b) Optionally, the reagents necessary for performing theamplification reaction.

[0249] In a preferred embodiment of the amplification kit describedabove, the primers are selected from the group consisting of thenucleotide sequences of SEQ ID Nos 72-101 and P1-P50

[0250] In another embodiment of the above amplification kit, theamplification product is detected by hybridization with a labeled probehaving a sequence which is complementary to the amplified region.

Biallelic Markers of RBP-7

[0251] The inventors have discovered nucleotide polymorphisms locatedwithin the genomic DNA containing the RBP-7 gene, and among them “SingleNucleotide Polymorphisms” or SNPs that are also termed biallelicmarkers.

[0252] The invention also relates to a nucleotide sequence, preferably apurified and/or isolated polynucleotide comprising a sequence defining abiallelic marker located in the sequence of a RBP-7 gene, a fragment orvariant thereof or a sequence complementary thereto. The sequencesdefining a biallelic marker may be of any length consistent with theirintended use, provided that they contain a polymorphic base from abiallelic marker. Preferably, the sequences defining a biallelic markerinclude the polymorphic base of one of SEQ ID Nos 30-71 or the sequencecomplementary thereto. In some embodiments the sequences defining abiallelic marker comprise one of the sequences selected from the groupconsisting of SEQ ID Nos 30-71 or the sequences complementary thereto.

[0253] In a preferred embodiment, the invention relates to a set ofpurified and/or isolated nucleotide sequences, each sequence comprisinga sequence defining a biallelic marker located in the sequence of aRBP-7 gene, wherein the set is characterized in that between about 30and 100%, preferably between about 40 and 60%, more preferably between50 and 60%, of the sequences defining a biallelic marker are selectedfrom the group consisting of SEQ ID Nos 30-71, the sequencescomplementary thereto, or a fragment or van ant thereof.

[0254] The invention further concerns a nucleic acid encoding a RBP-7protein, wherein said nucleic acid comprises a nucleotide sequenceselected from the group consisting of SEQ ID Nos 30-71 or the sequencescomplementary thereto.

[0255] The invention also relates to nucleotide sequence selected fromthe group consisting of SEQ ID Nos 30-71, the sequences complementarythereto, or a fragment or a variant thereof

[0256] A) Identification of Biallelic Markers

[0257] There are two preferred methods through which the biallelicmarkers of the present invention can be generated.

[0258] In a first method, DNA samples from unrelated individuals arepooled together, following which the genomic DNA of interest isamplified and sequenced. The nucleotide sequences thus obtained are thenanalyzed to identify significant polymorphisms. One of the majoradvantages of this method resides in the fact that the pooling of theDNA samples substantially reduces the number of DNA amplificationreactions and sequencing reactions which must be carried out Moreover,this method is sufficiently sensitive so that a biallelic markerobtained therewith usually shows a sufficient degree of informativenessfor conducting association studies.

[0259] In a second method for generating biallelic markers, the DNAsamples are not pooled and are therefore amplified and sequencedindividually. The resulting nucleotide sequences obtained are then alsoanalyzed to identify significant polymorphisms.

[0260] It will readily be appreciated that when this second method isused, a substantially higher number of DNA amplification reactions andsequencing reactions must be carried out. Moreover, a biallelic markerobtained using this method may show a lower degree of informativenessfor conducting association studies, e.g. if the frequency of its lessfrequent allele may be less than about 10%. It will further beappreciated that including such less informative biallelic markers inassociation studies to identify potential genetic associations with atrait may allow in some cases the direct identification of causalmutations, which may, depending on their penetrance, be rare mutations.This method is usually preferred when biallelic markers need to beidentified in order to perform association studies within candidategenes.

[0261] The following is a description of the various parameters of apreferred method used by the inventors to generate the markers of thepresent invention

[0262] 1— DNA Extraction

[0263] The genomic DNA samples from which the biallelic markers of thepresent invention are generated are preferably obtained from unrelatedindividuals corresponding to a heterogeneous population of known ethnicbackground.

[0264] The term “individual” as used herein refers to vertebrates,particularly members of the mammalian species and includes but is notlimited to domestic animals, sports animals, laboratory animals,primates and humans. Preferably, the individual is a human

[0265] The number of individuals from whom DNA samples are obtained canvary substantially, preferably from about 10 to about 1000, preferablyfrom about 50 to about 200 individuals. It is usually preferred tocollect DNA samples from at least about 100 individuals in order to havesufficient polymorphic diversity in a given population to identify asmany markers as possible and to generate statistically significantresults

[0266] As for the source of the genomic DNA to be subjected to analysis,any test sample can be foreseen without any particular limitation. Thesetest samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like, biological fluids such as cellculture supernatants, fixed tissue specimens including tumor andnon-tumor tissue and lymph node tissues; bone marrow aspirates and fixedcell specimens. The preferred source of genomic DNA used in the contextof the present invention is from peripheral venous blood of each donor.

[0267] The techniques of DNA extraction are well-known to the skilledtechnician. Details of a preferred embodiment are provided in Example 2.

[0268] Once genomic DNA from every individual in the given populationhas been extracted, it is preferred that a fraction of each DNA sampleis separated, after which a pool of DNA is constituted by assemblingequivalent amounts of the separated fractions into a single one.However, the person skilled in the art can choose to amplify the pooledor unpooled sequences

[0269] 2—DNA Amplification

[0270] The identification of biallelic markers in a sample of genomicDNA may be facilitated through the use of DNA amplification methods. DNAsamples can be pooled or unpooled for the amplification step. DNAamplification techniques are well known to those skilled in the art.Various methods to amplify DNA fragments carrying biallelic markers arefurther described hereinbefore in “Amplification of the RBP-7 gene”. ThePCR technology is the preferred amplification technique used to identifynew biallelic markers. A typical example of a PCR reaction suitable forthe purposes of the present invention is provided in Example 3.

[0271] In this context, one of the groups of oligonucleotides accordingto the present invention is a group of primers useful for theamplification of a genomic sequence encoding RBP-7 The primers pairs arecharacterized in that they have sufficient complementarity with anysequence of a strand of the RBP-7 gene to be amplified, preferably witha sequence of introns adjacent to exons to amplify, with regions of the3′ and 5′ ends of the RBP-7 gene, with splice sites or with 5′ UTRs or3′ UTRs to hybridize therewith.

[0272] These primers focus on exons and splice sites of the RBP-7 genesince an identified biallelic marker as described below presents ahigher probability to be an eventual causal mutation if it is located inthese functional regions of the gene

[0273] 15 pairs of primers were designed with the aim of amplifying eachof the 24 exons of the RBP-7 gene (Table 1) To these primers can beadded, at either end thereof, a further polynucleotide useful forsequencing such as described in Example 3. Preferred primers includethose having the nucleotide sequences disclosed in Example 3. Some ofthe primers according to the invention allow the amplification of themajority of the RBP-7 Exons shown in FIG. 2

[0274] The primers described above are individually useful asoligonucleotide probes in order to detect the corresponding RBP-7nucleotide sequence in a sample, and more preferably to detect thepresence of a RBP-7 DNA or RNA molecule in a sample suspected to containit

[0275] 3—Sequencing of Amplified Genomic DNA and Identification ofPolymorphisms

[0276] The amplification products generated as described above with theprimers of the invention are then sequenced using methods known andavailable to the skilled technician. Preferably, the amplified DNA issubjected to automated dideoxy terminator sequencing reactions using adye-primer cycle sequencing protocol

[0277] Following gel image analysis and DNA sequence extraction,sequence data are automatically processed with adequate software toassess sequence quality

[0278] The sequence data obtained as described above are transferred toa database, where quality control and validation steps are performed Abase-caller, working using a Unix system automatically flags suspectpeaks, taking into account the shape of the peaks, the inter-peakresolution, and the noise level The base-caller also performs anautomatic trimming. Any stretch of 25 or fewer bases having more than 4suspect peaks is usually considered unreliable and is discarded.

[0279] After this first sequence quality analysis, polymorphism analysissoftware is used to detect the presence of biallelic sites amongindividual or pooled amplified fragment sequences. The polymorphismsearch is based on the presence of superimposed peaks in theelectrophoresis pattern. These peaks, which present two distinct colors,correspond to two different nucleotides at the same position on thesequence. In order for peaks to be considered significant, peak heighthas to satisfy conditions of ratio between the peaks and conditions ofratio between a given peak and the surrounding peaks of the same color.

[0280] However, since the presence of two peaks can be an artifact dueto background noise, two controls are utilized to exclude theseartifacts:

[0281] the two DNA strands are sequenced and a comparison between thepeaks is carried out. The polymorphism has to be detected on bothstrands for validation.

[0282] all the sequencing electrophoresis patterns of the sameamplification product provided from distinct pools and/or individualsare compared. The homogeneity and the ratio of homozygous andheterozygous peak height are controlled through these distinct DNAs.

[0283] The detection limit for the frequency of biallelic polymorphismsdetected by sequencing pools of 100 individuals is about 0.1 for theminor allele, as verified by sequencing pools of known allelicfrequencies However, more than 90% of the biallelic polymorphismsdetected by the pooling method have a frequency for the minor allelehigher than 0.25. Therefore, the biallelic markers selected by thismethod have a frequency of at least 0 1 for the minor allele and lessthan 0.9 for the major allele, preferably at least 0 2 for the minorallele and less than 0 8 for the major allele, more preferably at least0.3 for the minor allele and less than 0.7 for the major allele, thus aheterozygosity rate higher than 0 18, preferably higher than 0.32, morepreferably higher than 0.42.

[0284] In a particular embodiment of the invention, the test samples area pool of 100 individuals and 50 individual samples. This is themethodology used in the preferred embodiment of the present invention,in which 21 biallelic markers have been identified in a genomic regioncontaining the RBP-7 gene Their location on the genomic RBP-7 DNA isshown in FIG. 2 and their particular sequences are disclosed in example4. The 24 exons and the intronic sequences surrounding the exons wereanalyzed. Among the 21 biallelic markers identified within the RBP-7gene, 6 biallelic markers are located with 4 different exons, and 15biallelic markers are located within the different intronic regions Thebiallelic markers 5-130-257, 5-143-84 and 5-143-101 respectively changeasparagine into glycine, glycine into glutainic acid and leucine intomethionine in the RBP-7 protein. The amino acid changes caused by the5-143-84 biallelic marker may be important for the RBP-7 biologicalactivity, since a neutral amino acid is replaced by a positively chargedamino acid in a RBP-7 region likely to contain a domain involved in anon-covalent interaction with the retinoblastoma protein or also a pRbrelated protein such as p107 or p130.

[0285] 4—Validation of the Biallelic Markers of the Present Invention

[0286] The polymorphisms are evaluated for their usefulness as geneticmarkers by validating that both alleles are present in a population.Validation of the biallelic markers is accomplished by genotyping agroup of individuals by a method of the invention and demonstrating thatboth alleles are present. Microsequencing is a preferred method ofgenotyping alleles. The validation by genotyping step may be performedon individual samples derived from each individual in the group or bygenotyping a pooled sample derived from more than one individual Thegroup can be as small as one individual if that individual isheterozygous for the allele in question. Preferably the group containsat least three individuals, more preferably the group contains five orsix individuals, so that a single validation test will be more likely toresult in the validation of more of the biallelic markers that are beingtested. It should be noted, however, that when the validation test isperformed on a small group it may result in a false negative result ifas a result of sampling error none of the individuals tested carries oneof the two alleles. Thus, the validation process is less useful indemonstrating that a particular initial result is an artifact, than itis at demonstrating that there is a bona fide biallelic marker at aparticular position in a sequence. All of the genotyping, haplotyping,and association study methods of the invention may optionally beperformed solely with validated biallelic markers

[0287] 5—Evaluation of the Frequency of the Biallelic Markers of thePresent Invention

[0288] The validated biallelic markers are further evaluated for theirusefulness as genetic markers by determining the frequency of the leastcommon allele at the biallelic marker site. The higher the frequency ofthe less common allele the greater the usefulness of the biallelicmarker in association and interaction studies. The determination of theleast common allele is accomplished by genotyping a group of individualsby a method of the invention and demonstrating that both alleles arepresent. This determination of frequency by genotyping step may beperformed on individual samples derived from each individual in thegroup or by genotyping a pooled sample derived from more than oneindividual. The group must be large enough to be representative of thepopulation as a whole. Preferably the group contains at least 20individuals, more preferably the group contains at least 50 individuals,most preferably the group contains at least 100 individuals. Of coursethe larger the group the greater the accuracy of the frequencydetermination because of reduced sampling error A biallelic markerwherein the frequency of the less common allele is 30% or more is termeda “high quality biallelic marker.” All of the genotyping, haplotyping,and association interaction study methods of the invention mayoptionally be performed solely with high quality biallelic markers.

[0289] B—Genotyping an Individual for Biallelic Markers

[0290] Methods are provided to genotype a biological sample for one ormore biallelic markers of the present invention, all of which may beperformed in vitro Such methods of genotyping comprise determining theidentity of a nucleotide at an RBP-7 biallelic marker site by any methodknown in the art These methods find use in genotyping case-controlpopulations in association studies as well as individuals in the contextof detection of alleles of biallelic markers which are known to beassociated with a given trait, in which case both copies of thebiallelic marker present in individual's genome are determined so thatan individual may be classified as homozygous or heterozygous for aparticular allele

[0291] These genotyping methods can be performed nucleic acid samplesderived from a single individual or pooled DNA samples.

[0292] Genotyping can be performed using similar methods as thosedescribed above for the identification of the biallelic markers, orusing other genotyping methods such as those further described below. Inpreferred embodiments, the comparison of sequences of amplified genomicfragments from different individuals is used to identify new biallelicmarkers whereas microsequencing is used for genotyping known biallelicmarkers in diagnostic and association study applications.

[0293] 1—Source of DNA for Genotyping

[0294] Any source of nucleic acids, in purified or non-purified form,can be utilized as the starting nucleic acid, provided it contains or issuspected of containing the specific nucleic acid sequence desired. DNAor RNA may be extracted from cells, tissues, body fluids and the like asdescribed above in “DNA extraction”. While nucleic acids for use in thegenotyping methods of the invention can be derived from any mammaliansource, the test subjects and individuals from which nucleic acidsamples are taken are generally understood to be human.

[0295] 2—Amplification of DNA Fragments Comprising Biallelic Markers

[0296] Methods and polynucleotides are provided to amplify a segment ofnucleotides comprising one or more biallelic marker of the presentinvention. It will be appreciated that amplification of DNA fragmentscomprising biallelic markers may be used in various methods and forvarious purposes and is not restricted to genotyping Nevertheless, manygenotyping methods, although not all, require the previous amplificationof the DNA region carrying the biallelic marker of interest. Suchmethods specifically increase the concentration or total number ofsequences that span the biallelic marker or include that site andsequences located either distal or proximal to it. Diagnostic assays mayalso rely on amplification of DNA segments carrying a biallelic markerof the present invention

[0297] Amplification of DNA may be achieved by any method known in theart. Amplification techniques are described above under the headings“Amplification of the RBP-7 gene”.

[0298] Some of these amplification methods are particularly suited forthe detection of single nucleotide polymorphisms and allow thesimultaneous amplification of a target sequence and the identificationof the polymorphic nucleotide as it is further described below

[0299] The identification of biallelic markers as described above allowsthe design of appropriate oligonucleotides, which can be used as primersto amplify DNA fragments comprising the biallelic markers of the presentinvention. Amplification can be performed using the primers initiallyused to discover new biallelic markers which are described herein or anyset of primers allowing the amplification of a DNA fragment comprising abiallelic marker of the present invention.

[0300] In some embodiments the present invention provides primers foramplifying a DNA fragment containing one or more biallelic markers ofthe present invention. Preferred amplification primers are listed inExample 3. It will be appreciated that the primers listed are merelyexemplary and that any other set of primers which produce amplificationproducts containing one or more biallelic markers of the presentinvention

[0301] The spacing of the primers determines the length of the segmentto be amplified In the context of the present invention amplifiedsegments carrying biallelic markers can range in size from at leastabout 25 bp to 35 kbp. Amplification fragments from 25-3000 bp aretypical, fragments from 50-1000 bp are preferred and fragments from100-600 bp are highly preferred It will be appreciated thatamplification primers for the biallelic markers may be any sequencewhich allow the specific amplification of any DNA fragment carrying themarkers. Amplification primers may be labeled or immobilized on a solidsupport as described under the headings entitled “Oligonucleotide probesand primers”.

[0302] 3—Methods of Genotyping DNA Samples for Biallelic Markers

[0303] a—Sequencing Assays

[0304] The amplification products generated above with the primers ofthe invention can be sequenced using methods known and available to theskilled technician. Preferably, the amplified DNA is subjected toautomated dideoxy terminator sequencing reactions using a dye-primercycle sequencing protocol. A sequence analysis can allow theidentification of the base present at the polymorphic site.

[0305] b—Microsequencing Assays

[0306] In microsequencing methods, the nucleotide at a polymorphic sitein a target DNA is detected by a single nucleotide primer extensionreaction. This method involves appropriate microsequencing primerswhich, hybridize just upstream of the polymorphic base of interest inthe target nucleic acid A polymerase is used to specifically extend the3′ end of the primer with one single ddNTP (chain terminator)complementary to the nucleotide at the polymorphic site Next theidentity of the incorporated nucleotide is determined in any suitableway.

[0307] Typically, microsequencing reactions are carried out usingfluorescent ddNTPs and the extended microsequencing primers are analyzedby electrophoresis on ABI 377 sequencing machines to determine theidentity of the incorporated nucleotide as described in EP 412 883, thedisclosure of which is incorporated herein by reference in its entiretyAlternatively capillary electrophoresis can be used in order to processa higher number of assays simultaneously. An example of a typicalmicrosequencing procedure that can be used in the context of the presentinvention is provided in Example 5.

[0308] Different approaches can be used for the labeling and detectionof ddNTPs A homogeneous phase detection method based on fluorescenceresonance energy transfer has been described by Chen and Kwok (1997) andChen et al. (1997). In this method amplified genomic DNA fragmentscontaining polymorphic sites are incubated with a 5′-fluorescein-labeledprimer in the presence of allelic dye-labeled dideoxyribonucleosidetriphosphates and a modified Taq polymerase. The dye-labeled primer isextended one base by the dye-terminator specific for the allele presenton the template. At the end of the genotyping reaction, the fluorescenceintensities of the two dyes in the reaction mixture are analyzeddirectly without separation or purification. All these steps can beperformed in the same tube and the fluorescence changes can be monitoredin real time.

[0309] Microsequencing may be achieved by the establishedmicrosequencing method or by developments or derivatives thereofAlternative methods include several solid-phase microsequencingtechniques. The basic microsequencing protocol is the same as describedpreviously, except that the method is conducted as a heterogenous phaseassay, in which the primer or the target molecule is immobilized orcaptured onto a solid support. To simplify the primer separation and theterminal nucleotide addition analysis, oligonucleotides are attached tosolid supports or are modified in such ways that permit affinityseparation as well as polymerase extension. The 5′ ends and internalnucleotides of synthetic oligonucleotides can be modified in a number ofdifferent ways to permit different affinity separation approaches, e.g.,biotinylation. If a single affinity group is used on theoligonucleotides, the oligonucleotides can be separated from theincorporated terminator regent. Thus eliminates the need of physical orsize separation. More than one oligonucleotide can be separated from theterminator reagent and analyzed simultaneously if more than one affinitygroup is used. This permits the analysis of several nucleic acid speciesor more nucleic acid sequence information per extension reaction. Theaffinity group need not be on the priming oligonucleotide but couldalternatively be present on the template. For example, immobilizationcan be carried out via an interaction between biotinylated DNA andstreptavidin-coated microtitration wells or avidin-coated polystyreneparticles In the same manner oligonucleotides or templates may beattached to a solid support in a high-density format In such solid phasemicrosequencing reactions, incorporated ddNTPs can be radiolabeled(Syvanen, 1994) or linked to fluorescein (Livak and Hainer, 1994). Thedetection of radiolabeled ddNTPs can be achieved throughscintillation-based techniques. The detection of fluorescein-linkedddNTPs can be based on the binding of antifluorescein antibodyconjugated with alkaline phosphatase, followed by incubation with achromogenic substrate (such as p-nitrophenyl phosphate). Other possiblereporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP)and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) orbiotinylated ddNTP and horseradish peroxidase-conjugated streptavidinwith o-phenylenediamine as a substrate (WO 92/15712, the disclosure ofwhich is incorporated herein by reference in its entirety) As yetanother alternative solid-phase microsequencing procedure, Nyren et al(1993) described a method relying on the detection of DNA polymeraseactivity by an enzymatic luminometric inorganic pyrophosphate detectionassay (ELIDA)

[0310] Pastinen et al. (1997) describe a method for multiplex detectionof single nucleotide polymorphism in which the solid phaseminisequencing principle is applied to an oligonucleotide array format.High-density arrays of DNA probes attached to a solid support (DNAchips) are further described below.

[0311] In one aspect the present invention provides polynucleotides andmethods to genotype one or more biallelic markers of the presentinvention by performing a microsequencing assay. Preferredmicrosequencing primers include those being featured in Example 5. Itwill be appreciated that the microsequencing primers listed in Example 5are merely exemplary and that, any primer having a 3′ end immediatelyadjacent to the polymorphic nucleotide may be used. Similarly, it willbe appreciated that microsequencing analysis may be performed for anybiallelic marker or any combination of biallelic markers of the presentinvention. One aspect of the present invention is a solid support whichincludes one or more microsequencing primers listed in Example 5, orfragments comprising at least 8, at least 12, at least 15, or at least20 consecutive nucleotides thereof and having a 3′ terminus immediatelyupstream of the corresponding biallelic marker, for determining theidentity of a nucleotide at a biallelic marker site.

[0312] c—Mismatch Detection Assays Based on Polymerases and Ligases

[0313] In one aspect the present invention provides polynucleotides andmethods to determine the allele of one or more biallelic markers of thepresent invention in a biological sample, by allele-specificamplification assays. Methods, primers and various parameters to amplifyDNA fragments comprising biallelic markers of the present invention arefurther described above.

[0314] Allele Specific Amplification Primers

[0315] Discrimination between the two alleles of a biallelic marker canalso be achieved by allele specific amplification, a selective strategy,whereby one of the alleles is amplified without amplification of theother allele This can be accomplished by placing the polymorphic base atthe 3′ end of one of the amplification primers. Because the extensionforms from the 3′ end of the primer, a mismatch at or near this positionhas an inhibitory effect on amplification. Therefore, under appropriateamplification conditions, these primers only direct amplification ontheir complementary allele. Determining the precise location of themismatch and the corresponding assay conditions are well within theordinary skill in the art

[0316] Ligation Amplification Based Methods

[0317] The “Oligonucleotide Ligation Assay” (OLA) uses twooligonucleotides which are designed to be capable of hybridizing toabutting sequences of a single strand of a target molecules One of theoligonucleotides is biotinylated, and the other is detectably labeled Ifthe precise complementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate that can be captured and detected. OLA is capableof detecting single nucleotide polymorphisms and may be advantageouslycombined with PCR as described by Nickerson et al. (1990). In thismethod, PCR is used to achieve the exponential amplification of targetDNA, which is then detected using OLA.

[0318] Other amplification methods which are particularly suited for thedetection of single nucleotide polymorphism include LCR (ligase chainreaction), Gap LCR (GLCR) which are described above in “Amplification ofthe RBP-7 gene”. LCR uses two pairs of probes to exponentially amplify aspecific target. The sequences of each pair of oligonucleotides, isselected to permit the pair to hybridize to abutting sequences of thesame strand of the target. Such hybridization forms a substrate for atemplate-dependant ligase. In accordance with the present invention, LCRcan be performed with oligonucleotides having the proximal and distalsequences of the same strand of a biallelic marker site. In oneembodiment, either oligonucleotide will be designed to include thebiallelic marker site. In such an embodiment, the reaction conditionsare selected such that the oligonucleotides can be ligated together onlyif the target molecule either contains or lacks the specific nucleotidethat is complementary to the biallelic marker on the oligonucleotide. Inan alternative embodiment, the oligonucleotides will not include thebiallelic marker, such that when they hybridize to the target molecule,a “gap” is created as described in WO 90/01069, the disclosure of whichis incorporated herein by reference in its entirety. This gap is then“filled” with complementary dNTPs (as mediated by DNA polymerase), or byan additional pair of oligonucleotides. Thus at the end of each cycle,each single strand has a complement capable of serving as a targetduring the next cycle and exponential allele-specific amplification ofthe desired sequence is obtained.

[0319] Ligase/Polymerase-mediated Genetic Bit Analysis™ is anothermethod for determining the identity of a nucleotide at a preselectedsite in a nucleic acid molecule (WO 95/21271, the disclosure of which isincorporated herein by reference in its entirety) This method involvesthe incorporation of a nucleoside triphosphate that is complementary tothe nucleotide present at the preselected site onto the terminus of aprimer molecule, and their subsequent ligation to a secondoligonucleotide. The reaction is monitored by detecting a specific labelattached to the reaction's solid phase or by detection in solution.

[0320] d—Hybridization Assay Methods

[0321] A preferred method of determining the identity of the nucleotidepresent at a biallelic marker site involves nucleic acid hybridization.The hybridization probes, which can be conveniently used in suchreactions, preferably include the probes defined herein. Anyhybridization assay may be used including Southern hybridization,Northern hybridization, dot blot hybridization and solid-phasehybridization (see Sambrook et al, 1989).

[0322] Hybridization refers to the formation of a duplex structure bytwo single stranded nucleic acids due to complementary base paringHybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch Specific probes can be designed that hybridize to one form of abiallelic marker and not to the other and therefore are able todiscriminate between different allelic forms. Allele-specific probes areoften used in pairs, one member of a pair showing perfect match to atarget sequence containing the original allele and the other showing aperfect match to the target sequence containing the alternative allele.Hybridization conditions should be sufficiently stringent that there isa significant difference in hybridization intensity between alleles, andpreferably an essentially binary response, whereby a probe hybridizes toonly one of the alleles. Stringent, sequence specific hybridizationconditions, under which a probe will hybridize only to the exactlycomplementary target sequence are well known in the art (Sambrook etal., 1989). Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. Althoughsuch hybridizations can be performed in solution, it is preferred toemploy a solid-phase hybridization assay. The target DNA comprising abiallelic marker of the present invention may be amplified prior to thehybridization reaction. The presence of a specific allele in the sampleis determined by detecting the presence or the absence of stable hybridduplexes formed between the probe and the target DNA. The detection ofhybrid duplexes can be carried out by a number of methods. Variousdetection assay formats are well known which utilize detectable labelsbound to either the target or the probe to enable detection of thehybrid duplexes. Typically, hybridization duplexes are separated fromunhybridized nucleic acids and the labels bound to the duplexes are thendetected. Those skilled in the art will recognize that wash steps may beemployed to wash away excess target DNA or probe as well as unboundconjugate. Further, standard heterogeneous assay formats are suitablefor detecting the hybrids using the labels present on the primers andprobes Preferably, the hybrids can be bound to a solid phase reagent byvirtue of a capture label and detected by virtue of a detection label.In cases where the detection label is directly detectable, the presenceof the hybrids on the solid phase can be detected by causing the labelto produce a detectable signal, if necessary, and detecting the signal.In cases where the label is not directly detectable, the capturedhybrids can be contacted with a conjugate, which generally comprises abinding member attached to a directly detectable label The conjugatebecomes bound to the complexes and the conjugates presence on thecomplexes can be detected with the directly detectable label. Thus, thepresence of the hybrids on the solid phase reagent can be determined.

[0323] The polynucleotides provided herein can be used to produce probeswhich can be used in hybridization assays for the detection of biallelicmarker alleles in biological samples. These probes are characterized inthat they preferably comprise between 8 and 50 nucleotides, and in thatthey are sufficiently complementary to a sequence comprising a biallelicmarker of the present invention to hybridize thereto and preferablysufficiently specific to be able to discrimnate the targeted sequencefor only one nucleotide variation. A particularly preferred probe is 25nucleotides in length. Preferably the polymorphic site of the biallelicmarker is within 4 nucleotides of the center of the polynucleotideprobe. In particularly preferred probes the polymorphic site of thebiallelic marker is at the center of said polynucleotide.

[0324] Preferably the probes of the present invention are labeled orimmobilized on a solid support Labels and solid supports are furtherdescribed in “Oligonucleotide probes and primers”. Detection probes aregenerally nucleic acid sequences or uncharged nucleic acid analogs suchas, for example peptide nucleic acids which are disclosed inInternational Patent Application WO 92/20702, morpholino analogs whichare described in U.S. Pat. Nos. 5,185,444; 5,034,506 and 5,142,047, thedisclosures of which are incorporated herein by reference in theirentireties. The probe may have to be rendered “non-extendable” in thatadditional dNTPs cannot be added to the probe. In and of themselvesanalogs usually are non-extendable and nucleic acid probes can berendered non-extendable by modifying the 3′ end of the probe such thatthe hydroxyl group is No. longer capable of participating in elongation.For example, the 3′ end of the probe can be functionalized with thecapture or detection label to thereby consume or otherwise block thehydroxyl group. Alternatively, the 3′ hydroxyl group simply can becleaved, replaced or modified, U.S. patent application Ser. No.07/049,061 filed Apr. 19, 1993 describes modifications, which can beused to render a probe non-extendable.

[0325] The probes of the present invention are useful for a number ofpurposes. By assaying the hybridization to an allele specific probe, onecan detect the presence or absence of a biallelic marker allele in agiven sample.

[0326] High-Throughput parallel hybridizations in array format arespecifically encompassed within “hybridization assays” and are describedbelow.

[0327] e—Hybridization to Addressable Arrays of Oligonucleotides

[0328] DNA chips result from the adaptation of computer chips to biologyEfficient access to polymorphism information is obtained through a basicstructure comprising high-density arrays of oligonucleotide probesattached to a solid support (the chip) at selected positions. Each DNAchip can contain thousands to millions of individual synthetic DNAprobes arranged in a grid-like pattern and miniaturized to the size of adime.

[0329] The chip technology has already been applied with success innumerous cases. For example, the screening of mutations has beenundertaken in the BRCA1 gene, in S cerevisiae mutant strains, and in theprotease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al.,1996; Kozal et al., 1996). Chips of various formats for use in detectingbiallelic polymorphisms can be produced on a customized basis byAffymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and ProtogeneLaboratories.

[0330] In general, these methods employ arrays of oligonucleotide probesthat are complementary to target nucleic acid sequence segments from anindividual which, target sequences include a polymorphic marker.EP785280, the disclosure of which is incorporated herein by reference inits entirety, describes a tiling strategy for the detection of singlenucleotide polymorphisms. Briefly, arrays may generally be “tiled” for alarge number of specific polymorphisms. By “tiling” is generally meantthe synthesis of a defined set of oligonucleotide probes which is madeup of a sequence complementary to the target sequence of interest, aswell as preselected variations of that sequence, e g., substitution ofone or more given positions with one or more members of the basis set ofmonomers, i.e. nucleotides. Tiling strategies are further described inPCT Application No. WO 95/11995, the disclosure of which is incorporatedherein by reference in its entirety In a particular aspect, arrays aretiled for a number of specific, identified biallelic marker sequences.In particular the array is tiled to include a number of detectionblocks, each detection block being specific for a specific biallelicmarker or a set of biallelic markers For example, a detection block maybe tiled to include a number of probes, which span the sequence segmentthat includes a specific polymorphism. To ensure probes that arecomplementary to each allele, the probes are synthesized in pairsdiffering at the biallelic marker. In addition to the probes differingat the polymorphic base, monosubstituted probes are also generally tiledwithin the detection block These monosubstituted probes have bases atand up to a certain number of bases in either direction from thepolymorphism, substituted with the remaining nucleotides (selected fromA, T, G, C and U). Typically the probes in a tiled detection block willinclude substitutions of the sequence positions up to and includingthose that are 5 bases away from the polymorphic site of the biallelicmarker. The monosubstituted probes provide internal controls for thetiled array, to distinguish actual hybridization from artifactualcross-hybridization Upon completion of hybridization with the targetsequence and washing of the array, the array is scanned to determine theposition on the array to which the target sequence hybridizes. Thehybridization data from the scanned array is then analyzed to identifywhich allele or alleles of the biallelic marker are present in thesample Hybridization and scanning may be carried out as described in PCTApplication No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186,the disclosures of which are incorporated herein by reference in theirentireties.

[0331] Thus, in some embodiments, the chips may comprise an array ofnucleic acid sequences of fragments of about 15 nucleotides in length.In further embodiments, the chip may comprise an array including atleast one of the sequences selected from the group consisting of thenucleic acids of the sequences set forth as SEQ ID Nos 30-75 and thesequences complementary thereto, or a fragment thereof at least about 8consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30,or 40 consecutive nucleotides comprising a biallelic marker of thepresent invention. In some embodiments, the chip may comprise an arrayof at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of theinvention. Solid supports and polynucleotides of the present inventionattached to solid supports are further described in “Oligonucleotideprimers and probes”

[0332] f—Integrated Microsequencing and Capillary Electrophoresis Chips

[0333] Another technique, which may be used to analyze polymorphisms,includes multicomponent integrated systems, which miniaturize andcompartmentalize processes such as PCR and capillary electrophoresisreactions in a single functional device An example of such technique isdisclosed in U.S. Pat. No. 5,589,136, the disclosure of which isincorporated herein by reference in its entirety, which describes theintegration of PCR amplification and capillary electrophoresis in chips

[0334] Integrated systems can be envisaged mainly when microfluidicsystems are used These systems comprise a pattern of microchannelsdesigned onto a glass, silicon, quartz, or plastic wafer included on amicrochip. The movements of the samples are controlled by electric,electroosmotic or hydrostatic forces applied across different areas ofthe microchip to create functional microscopic valves and pumps with nomoving parts.

[0335] For genotyping biallelic markers, the microfluidic system mayintegrate nucleic acid amplification, microsequencing, capillaryelectrophoresis and a detection method such as laser-inducedfluorescence detection.

Association Studies with the Biallelic Markers of the RBP-7 Gene

[0336] The identification of genes involved in suspected heterogeneous,polygenic and multifactorial traits such as cancer can be carried outthrough two main strategies currently used for genetic mapping linkageanalysis and association studies. Association studies examine thefrequency of marker alleles in unrelated trait positive (T+) individualscompared with trait negative (T−) controls, and are generally employedin the detection of polygenic inheritance. Association studies as amethod of mapping genetic traits rely on the phenomenon of linkagedisequilibrium, which is described below.

[0337] If two genetic loci lie on the same chromosome, then sets ofalleles of these loci on the same chromosomal segment (calledhaplotypes) tend to be transmitted as a block from generation togeneration. When not broken up by recombination, haplotypes can betracked not only through pedigrees but also through populations. Theresulting phenomenon at the population level is that the occurrence ofpairs of specific alleles at different loci on the same chromosome isnot random, and the deviation from random is called linkagedisequilibrium (LD).

[0338] If a specific allele in a given gene is directly involved incausing a particular trait T, its frequency will be statisticallyincreased in a T+ population when compared to the frequency in a T−population. As a consequence of the existence of LD, the frequency ofall other alleles present in the haplotype carrying the trait-causingallele (TCA) will also be increased in T+ individuals compared to T−individuals. Therefore, association between the trait and any allele inlinkage disequilibrium with the trait-causing allele will suffice tosuggest the presence of a trait-related gene in that particular allele'sregion. Linkage disequilibrium allows the relative frequencies in T+ andT− populations of a limited number of genetic polymorphisms(specifically biallelic markers) to be analyzed as an alternative toscreening all possible functional polymorphisms in order to findtrait-causing alleles

[0339] The general strategy to perform association studies usingbiallelic markers derived from a candidate region is to scan two groupsof individuals (trait+ and trait− control individuals which arecharacterized by a well defined phenotype as described below) in orderto measure and statistically compare the allele frequencies of suchbiallelic markers in both groups.

[0340] If a statistically significant association with a trait isidentified for at least one or more of the analyzed biallelic markers,one can assume that: either the associated allele is directlyresponsible for causing the trait (associated allele is the TCA), or theassociated allele is in LD with the TCA If the evidence indicates thatthe associated allele within the candidate region is most probably notthe TCA but is in LD with the real TCA, then the TCA, and by consequencethe gene carrying the TCA, can be found by sequencing the vicinity ofthe associated marker.

[0341] It is another object of the present invention to provide a methodfor the identification and characterization of an association betweenalleles for one or several biallelic markers of the human RBP-7 gene anda trait The method comprises the steps of:

[0342] genotyping a marker or a group of biallelic markers according tothe invention in trait positive and trait negative individuals; and

[0343] establishing a statistically significant association between oneallele of at least one marker and the trait

[0344] Preferably, the trait positive and trait negative individuals areselected from non-overlapping phenotypes, at opposite ends of thenon-bimodal phenotype spectra of the trait under study. In someembodiments, the biallelic marker is one of the biallelic markers of thepresent invention.

[0345] In a preferred embodiment, the trait is a disease and preferablya cancer.

[0346] The present invention also provides a method for theidentification and characterization of an association between ahaplotype comprising alleles for several biallelic markers of the humanRBP-7 gene and a trait. The method comprises the steps of:

[0347] genotyping a group of biallelic markers according to theinvention in trait positive and trait negative individuals; and

[0348] establishing a statistically significant association between ahaplotype and the trait.

[0349] In some embodiments, the haplotype comprises two or morebiallelic markers defined in SEQ ID Nos 30-71

[0350] The step of testing for and detecting the presence of DNAcomprising specific alleles of a biallelic marker or a group ofbiallelic markers of the present invention can be carried out asdescribed further below.

Vectors for the Expression of a Regulatory or a Coding PolynucleotideAccording to the Invention

[0351] Generally, a recombinant vector of the invention may comprise anyof the polynucleotides described herein, including regulatory sequences,coding sequences and polynucleotide constructs, as well as any RBP-7primer or probe as defined above. More particularly, the recombinantvectors of the present invention can comprise any of the polynucleotidesdescribed in the “RBP-7 Gene, Corresponding cDNAs And RBP-7 Coding AndRegulating Sequences” section, and the “Oligonucleotide Probes AndPrimers” section.

[0352] Any of the regulatory polynucleotides or the codingpolynucleotides of the invention may be inserted into recombinantvectors for expression in a recombinant host cell or a recombinant hostorganism

[0353] Thus, the present invention also encompasses a family ofrecombinant vectors that contains either a RBP-7 regulatorypolynucleotide or a RBP-7 coding polynucleotide or both of them.Preferably, the present invention concerns recombinant vectors thatcontains either a RBP-7 regulatory polynucleotide or a RBP-7 codingpolynucleotide comprising at least one of the biallelic markers of theinvention, particularly those of SEQ ID Nos 30-71.

[0354] More particularly, the present invention also relates toexpression vectors which include nucleic acids encoding a RBP-7 proteinunder the control of either a RBP-7 regulatory polynucleotide, or anexogenous regulatory sequence.

[0355] Another aspect of the present invention is a recombinantexpression vector comprising a nucleic acid selected from the groupconsisting of SEQ ID Nos 1, 4, 5-28 or complementary sequences theretoor fragments or van ants thereof.

[0356] Another preferred recombinant expression vector according to theinvention comprises a nucleic acid comprising a combination of at leasttwo polynucleotides selected from the group consisting of SEQ ID Nos5-28 or the sequences complementary thereto, wherein the polynucleotidesare arranged within the nucleic acid, from the 5′ end to the 3′ end ofsaid nucleic acid, in the same order than in the SEQ ID No. 1.

[0357] Another aspect of the invention is a recombinant expressionvector comprising a nucleic acid selected from the group consisting ofSEQ ID No. 2 or 3 or the sequences complementary thereto or abiologically active fragment or variant thereof.

[0358] A further aspect of the invention is a recombinant expressionvector comprising a purified or isolated nucleic acid comprising

[0359] a) a nucleic acid comprising the nucleotide sequence SEQ ID No.2, a fragment or variant thereof or a nucleotide sequence complementarythereto,

[0360] b) a polynucleotide encoding a protein or a polynucleotide ofinterest.

[0361] The invention also encompasses a recombinant expression vectorcontaining a polynucleotide comprising, consisting essentially of, orconsisting of

[0362] a) a nucleic acid comprising a regulatory polynucleotide of SEQID No. 2, or the sequence complementary thereto, or a biologicallyactive fragment or variant thereof, and

[0363] b) a polynucleotide encoding a polypeptide or a polynucleotide ofinterest.

[0364] c) Optionally, the expression vector may further comprise anucleic acid comprising a regulatory polynucleotide of SEQ ID No. 3, orthe sequence complementary thereto, or a biologically active fragment orvariant thereof.

[0365] The vector containing the appropriate DNA sequence as describedabove, more preferably a RBP-7 regulatory polynucleotide, a RBP-7 codingpolynucleotide or both of them, can be utilized to transform anappropriate host to allow the expression of the desired polypeptide orpolynucleotide

[0366] Vectors

[0367] A recombinant vector according to the invention comprises, but isnot limited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or evena linear DNA molecule which may comprise, consist essentially of, orconsist of a chromosomal, non-chromosomal and synthetic DNA. Such arecombinant vector can comprise a transcriptional unit comprising anassembly of

[0368] (1) a genetic element or elements having a regulatory role ingene expression, for example promoters or enhancers. Enhancers arecis-acting elements of DNA, usually from about 10 to 300 bp that act onthe promoter to increase the transcription.

[0369] (2) a structural or coding sequence which is transcribed intomRNA and eventually translated into a polypeptide, and

[0370] (3) appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems preferably include a leader sequence enablingextracellular secretion of translated protein by a host cellAlternatively, where recombinant protein is expressed without a leaderor transport sequence, it may include an N-terminal residue. Thisresidue may or may not be subsequently cleaved from the expressedrecombinant protein to provide a final product

[0371] Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The selectable markergenes can be for example dihydrofolate reductase or neomycin resistancefor eukaryotic cell culture, TRP1 for S cerevisiae or tetracycline,rifampicine or ampicillin resistance in E coli, or levan saccharase formycobacteria The heterologous structural sequence is assembled inappropriate phase with translation initiation and termination sequences,and preferably a leader sequence capable of directing secretion oftranslated protein into the periplasmic space or extracellular medium.

[0372] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired polypeptide withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host.

[0373] As a representative but non-limiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), andGEM1 (Promega Biotec, Madison, Wis., USA).

[0374] A suitable vector for the expression of the RBP-7 proteinabove-defined or their peptide fragments is a baculovirus vector thatcan be propagated in insect cells and in insect cell lines A specificsuitable host vector system is the pVL1392/1393 baculovirus transfervector (Pharmingen) that is used to transfect the SF9 cell line (ATCCNo. CRL 1711) which is derived from Spodoptera frugiperda. Otherbaculovirus vectors are described in Chai et al. (1993), Vlasak et al.(1983) and Lenhardt et al. (1996).

[0375] Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example SV40 origin, early promoter, enhancer, splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0376] Large numbers of suitable vectors and promoters are known tothose of skill in the art, and commercially available, such as bacterialvectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); or eukaryoticvectors: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV,pMSG, pSVL (Pharmacia); baculovirus transfer vector pVL1392/1393(Pharmingen); pQE-30 (QIAexpress).

[0377] Promoters

[0378] The suitable promoter regions used in the expression vectorsaccording to the present invention are choosen taking into account ofthe cell host in which the heterologous gene has to be expressed

[0379] Preferred bacterial promoters are the LacI, LacZ, the T3 or T7bacteriophage RNA polymerase promoters, the polyhedrin promoter, or thep10 protein promoter from baculovirus (Kit Novagen) (Smith et al.,1983.; O'Reilly et al., 1992), the lambda P_(R) promoter or also the trcpromoter.

[0380] Preferred promoters for the expression of the heterologous genein eukaryotic hosts are the early promoter of CMV, the Herpes simplexvirus thymidine kinase promoter, the early or the late promoter fromSV40, the LTR regions of certain retroviruses or also the mousemetallothionein 1 promoter.

[0381] Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors Particularly named bacterial promoters includelacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promotersinclude CMV immediate early, HSV thymidine kinase, early and late SV40,LTRs from retrovirus, and mouse metallothionein-L Selection of aconvenient vector and promoter is well within the level of ordinaryskill in the art

[0382] The choice of a determined promoter, among the above-describedpromoters is well in the ability of one skill in the art, guided by hisknowledge in the genetic engineering technical field, and by being alsoguided by the book of Sambrook et al. in 1989 or also by the proceduresdescribed by Fuller et al. in 1996.

[0383] Other Types Of Vectors

[0384] The in vivo expression of a RBP-7 polypeptide or a fragment or avariant thereof may be useful in order to study the physiologicalconsequences of a deregulation of its in vivo synthesis on thephysiology of the recipient recombinant host organism under study, moreparticularly on the cell differentiation and on an eventual abnormalproliferation of various kinds of cells, including T cells andepithelial cells.

[0385] Consequently, the present invention also relates to recombinantexpression vectors mainly designed for the in vivo production of atherapeutic peptide fragment by the introduction of the geneticinformation in the organism of the patient to be treated. This geneticinformation may be introduced in vitro in a cell that has beenpreviously extracted from the organism, the modified cell beingsubsequently reintroduced in the said organism, directly in vivo intothe appropriate tissue.

[0386] The method for delivering the corresponding protein or peptide tothe interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide into the interstitial space of a tissue comprising the cell,whereby the naked polynucleotide is taken up into the interior of thecell and has a physiological effect

[0387] In a specific embodiment, the invention provides a compositionfor the in vivo production of a RBP-7 polypeptide containing a nakedpolynucleotide operatively coding for a RBP-7 polypeptide or a fragmentor a variant thereof, in solution in a physiologically acceptablecarrier and suitable for introduction into a tissue to cause cells ofthe tissue to express the said protein or polypeptide.

[0388] Advantageously, the composition described above is administeredlocally, near the site in which the expression of a RBP-7 polypeptide ora fragment or a variant thereof is sought.

[0389] The polynucleotide operatively coding for a RBP-7 polypeptide ora fragment or variant thereof may be a vector comprising the genomic DNAor the complementary DNA (cDNA) coding for the corresponding protein orits protein derivative and a promoter sequence allowing the expressionof the genomic DNA or the complementary DNA in the desired eukaryoticcells, such as vertebrate cells, specifically mammalian cells.

[0390] The promoter contained in such a vector is selected among thegroup comprising

[0391] an internal or an endogenous promoter, such as the naturalpromoter associated with the structural gene coding for the desiredRBP-7 polypeptide or the fragment or variant thereof; such a promotermay be completed by a regulatory element derived from the vertebratehost, in particular an activator element;

[0392] a promoter derived from a cytoskeletal protein gene such as thedesmin promoter (Bolmont et al, 1990; Zhenlin et al., 1989).

[0393] As a general feature, the promoter may be heterologous to thevertebrate host, but it is advantageously homologous to the vertebratehost

[0394] By a promoter heterologous to the vertebrate host is intended apromoter that is not found naturally in the vertebrate host.

[0395] Compositions comprising a polynucleotide are described in the PCTApplication No. WO 90/11092 and also in the PCT Application No WO95/11307 as well as in the articles of Tacson et al. (1996) and ofHuygen et al. (1996), the disclosures of which are incorporated hereinby reference in their entireties.

[0396] In another embodiment, the DNA to be introduced is complexed withDEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda etal., 1989), with lipids (Felgner et al, 1987) or encapsulated withinliposomes (Fraley et al., 1980).

[0397] In another embodiment, the polynucleotide encoding a RBP-7polypeptide or a fragment or a variant thereof may be included in atransfection system comprising polypeptides that promote its penetrationwithin the host cells as it is described in the PCT Application WO95/10534, the disclosure of which is incorporated herein by reference inits entirety.

[0398] The vector according to the present invention may advantageouslybe administered in the form of a gel that facilitates their transfectioninto the cells. Such a gel composition may be a complex of poly-L-lysineand lactose, as described by Midoux (1993) or also poloxamer 407 asdescribed by Pastore (1994). Said vector may also be suspended in abuffer solution or be associated with liposomes.

[0399] The amount of the vector to be injected to the desired hostorganism vary according to the site of injection As an indicative dose,it will be injected between 0, 1 and 100 μg of the vector in an animalbody, preferably a mammal body, for example a mouse body.

[0400] In another embodiment of the vector according to the invention,said vector may be introduced in vitro in a host cell, preferably in ahost cell previously harvested from the animal to be treated and morepreferably a somatic cell such as a muscle cell. In a subsequent step,the cell that has been transformed with the vector coding for thedesired RBP-7 polypeptide or the desired fragment or variant thereof isimplanted back into the animal body in order to deliver the recombinantprotein within the body either locally or systemically.

[0401] Suitable vectors for the in vivo expression of a RBP-7polypeptide or a fragment or a variant thereof are described hereunder.

[0402] In one specific embodiment, the vector is derived from anadenovirus. Preferred adenovirus vectors according to the invention arethose described by Feldman and Steg (1996) or Ohno et al (1994). Anotherpreferred recombinant adenovirus according to this specific embodimentof the present invention is the adenovirus described by Ohwada et al.(1996) or the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or anadenovirus of animal origin (French Patent Application No. FR-93.05954,the disclosure of which is incorporated herein by reference in itsentirety).

[0403] Among the adenoviruses of animal origin it can be cited theadenoviruses of canine (CAV2, strain Manhattan or A26/61[ATCC VR-800]),bovine, murine (Mav1, Beard et al., 1980) or simian (SAV). Otheradenoviruses are described by Levrero et al. (1991), Graham et al(1984), in the European Patent Application No. EP-185.573 or in the PCTApplication No WO 95/14785, the disclosures of which are incorporatedherein by reference in their entireties.

[0404] Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery system ofchoice for the transfer of exogenous polynucleotides in vivo,particularly to mammals, including humans These vectors provideefficient delivery of genes into cells, and the transferred nucleicacids are stably integrated into the chromosomal DNA of the hostSuitable retroviruses used according to the present invention includethose described in the PCT Application No. WO 93/25234, the PCTApplication No. WO 94/06920, the PCT Application No WO 94/24298, Roth etal. (1996), Roux et al (1989), Juhan et al. (1992) and Neda et al(1991), the disclosures of which are incorporated herein by reference intheir entireties Other preferred retrovirus include Murine LeukemiaViruses such as 4070A and 1504A (Hartley et al., 1976), Abelson (ATCCNo. VR-999), Friend (ATCC No VR-245), Gross (ATCC No. VR-590), Rauscher(ATCC No. VR-998) and Moloney Munne Leukemia Virus (ATCC No. VR-190; PCTApplication No. WO 94/24298), the disclosure of which is incorporatedherein by reference in its entirety, and also Rous Sarcoma Viruses suchas Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728.

[0405] Yet another viral vector system that is contemplated by theinvention comprises the adeno-associated virus (AAV) Adeno-associatedvirus is a naturally occuring defective virus that requires anothervirus, such as an adenovirus or a herpes virus, as a helper virus forefficient replication and a productive life cycle (Muzyczka et al.,1992) It is also one of the few viruses that may integrate its DNA intonon-dividing cells, and exhibits a high frequency of stable integration(Flotte et al, 1992; Samulski et al., 1989; McLaughlin et al, 1989). Oneadvantageous feature of AAV derives from its reduced efficacy fortransducing primary cells relative to transformed cells.

[0406] Other compositions containing a vector of the invention compriseadvantageously an oligonucleotide fragment of the nucleic sequence ofRBP-7 as an antisense tool that inhibits the expression of thecorresponding gene and is thus useful to inhibit the expression of theRBP-7 gene in the tagged cells or organs. Preferred methods usingantisense polynucleotide according to the present invention are theprocedures described by Sczakiel et al. (1995) or also in the PCTApplication No. WO 95/24223, the disclosure of which is incorporatedherein by reference in its entirety

[0407] Vectors Suitable for Homologous Recombination

[0408] Other suitable vectors, particularly for the expression of genesin mammalian cells, may be selected from the group of vectors consistingof P1 bacteriophages, and bacterial artificial chromosomes (BACs). Thesetypes of vectors may contain large inserts ranging from about 80-90 kb(P1 bacteriophage) to about 300 kb (BACs). P1 bacteriophage.

[0409] The construction of P1 bacteriophage vectors such as p158 orp158/neo8 are notably described by Steinberg (1992, 1994). RecombinantP1 clones comprising RBP-7 nucleotide sequences may be designed forinserting large polynucleotides of more than 40 kb (Linton et al, 1993).To generate P1 DNA for transgenic experiments, a preferred protocol isthe protocol described by McCormick et al (1994). Briefly, E coli(preferably strain NS3529) harboring the P1 plasmid are grown overnightin a suitable broth medium containing 25 μg/ml of kanamycin. The P1 DNAis prepared from the E. coli by alkaline lysis using the Qiagen PlasmidMaxi kit (Qiagen, Chatsworth, Calif., USA), according to themanufacturer's instructions. The P1 DNA is purified from the bacteriallysate on two Qiagen-tip 500 columns, using the washing and elutionbuffers contained in the kit. A phenol/chloroform extraction is thenperformed before precipitating the DNA with 70% ethanol. Aftersolubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), theconcentration of the DNA is assessed by spectrophotometry

[0410] When the goal is to express a P1 clone comprising RBP-7nucleotide sequences in a transgenic animal, typically in transgenicmice, it is desirable to remove vector sequences from the P1 DNAfragment, for example by cleaving the P1 DNA at rare-cutting siteswithin the P1 polylinker (SfiI, NotI or SalI). The P1 insert is thenpurified from vector sequences on a pulsed-field agarose gel, usingmethods similar using methods similar to those originally reported forthe isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al.,1993). At this stage, the resulting purified insert DNA can beconcentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit(Millipore, Bedford, Mass., USA 30,000 molecular weight limit) and thendialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μMEDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on amicrodyalisis membrane (type VS, 0.025 μM from Millipore). Theintactness of the purified P1 DNA insert is assessed by electrophoresison 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel andstaining with ethidium bromide.

[0411] Bacterial Artificial Chromosomes (BACs)

[0412] The bacterial artificial chromosome (3AC) cloning system (Shizuyaet al., 1992) has been developed to stably maintain large fragments ofgenomic DNA (100-300 kb) in E coli. A preferred BAC vector is thepBeloBAC11 vector that has been described by Kim et al. (1996) BAClibraries are prepared with this vector using size-selected genomic DNAthat has been partially digested using enzymes that permit ligation intoeither the Bam HI or HindIII sites in the vector. Flanking these cloningsites are T7 and SP6 RNA polymerase transcription initiation sites thatcan be used to generate end probes by either RNA transcription or PCRmethods. After the construction of a BAC library in E coli, BAC DNA ispurified from the host cell as a supercooled circle. Converting thesecircular molecules into a linear form precedes both size determinationand introduction of the BACs into recipient cells. The cloning site isflanked by two Not I sites, permitting cloned segments to be excisedfrom the vector by Not I digestion. Alternatively, the DNA insertcontained in the pBeloBAC11 vector may be linearized by treatment of theBAC vector with the commercially available enzyme lambda terminase thatleads to the cleavage at the unique cosN site, but this cleavage methodresults in a full length BAC clone containing both the insert DNA andthe BAC sequences

[0413] Specific DNA Construct Vector for Homologous Recombination

[0414] The term “DNA construct” is understood to mean a linear orcircular purified or isolated polynucleotide that has been artificiallydesigned and which comprises at least two nucleotide sequences that arenot found as c contiguous nucleotide sequences in their naturalenvironment

DNA Construct that Enables Directing Temporal and Spatial GeneExpression in Recombinant Cell Hosts and in Transgenic Animals

[0415] In order to study the physiological and phenotype consequences ofa lack of synthesis of the RBP-7 protein, both at the cell level and atthe multi cellular organism level, in particular as regards to disordersrelated to abnormal cell proliferation, notably cancers, the inventionalso encompasses DNA constructs and recombinant vectors enabling aconditional expression of a specific allele of the RBP-7 genomicsequence or cDNA and also of a copy of this genomic sequence or cDNAharboring substitutions, deletions, or additions of one or more bases asregards to the RBP-7 nucleotide sequence of SEQ ID Nos 1 or 4, or afragment thereof, these base substitutions, deletions or additions beinglocated either in an exon, an intron or a regulatory sequence, butpreferably in the 5′-regulatory sequence or in an exon of the RBP-7genomic sequence or within the RBP-7 cDNA of SEQ ID No. 4.

[0416] A first preferred DNA construct is based on the tetracyclineresistance operon tet from E coli transposon Tn110 for controlling theRBP-7 gene expression, such as described by Gossen et al. (1992, 1995)and Furth et al. (1994). Such a DNA construct contains seven tetoperator sequences from Tn10 (tetop) that are fused to either a minimalpromoter or a 5′-regulatory sequence of the RBP-7 gene, said minimalpromoter or said RBP-7 regulatory sequence being operably linked to apolynucleotide of interest that codes either for a sense or an antisenseoligonucleotide or for a polypeptide, including a RBP-7 polypeptide or apeptide fragment thereof This DNA construct is functional as aconditional expression system for the nucleotide sequence of interestwhen the same cell also comprises a nucleotide sequence coding foreither the wild type (tTA) or the mutant (rTA) repressor fused to theactivating domain of viral protein VP16 of herpes simplex virus, placedunder the control of a promoter, such as the HCMVIE1 enhancer/promoteror the MMTV-LTR. Indeed, a preferred DNA construct of the invention willcomprise both the polynucleotide containing the tet operator sequencesand the polynucleotide containing a sequence coding for the tTA or therTA repressor

[0417] In the specific embodiment wherein the conditional expression DNAconstruct contains the sequence encoding the mutant tetracyclinerepressor rTA, the expression of the polynucleotide of interest issilent in the absence of tetracycline and induced in its presence

[0418] DNA Constructs Allowing Homologous Recombination: ReplacementVectors

[0419] A second preferred DNA construct will comprise, from 5′-end to3′-e nd: (a) a first nucleotide sequence that is comprised in the RBP-7genomic sequence, (b) a nucleotide sequence comprising a positiveselection marker, such as the marker for neomycine resistance (neo), and(c) a second nucleotide sequence that is comprised in the RBP-7 genomicsequence, and is located on the genome downstream the first RBP-7nucleotide sequence (a)

[0420] In a preferred embodiment, this DNA construct also comprises anegative selection marker located upstream the nucleotide sequence (a)or downstream the nucleotide sequence (b). Preferably, the negativeselection marker is the thymidine kinase (tk) gene (Thomas et al.,1986), the hygromycine beta gene (Te Riele et al, 1990), the hprt gene(Van der Lugt et al, 1991; Reid et al, 1990) or the Diphteria toxin Afragment (Dt-A) gene (Nada et al, 1993; Yagi et al. 1990) Preferably,the positive selection marker is located within a RBP-7 exon sequence soas to interrupt the sequence encoding a RBP-7 protein

[0421] These replacement vectors are described for example by Thomas etal. (1986; 1987), Mansour et al. (1988) and Koller et al. (1992).

[0422] The first and second nucleotide sequences (a) and (c) may beindifferently located within a RBP-7 regulatory sequence, an intronicsequence, an exon sequence or a sequence containing both regulatoryand/or intronic and/or exon sequences. The size of the nucleotidesequences (a) and (c) is ranging from 1 to 50 kb, preferably from 1 to10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4kb.

[0423] DNA Constructs Allowing Homologous Recombination: Cre-Loxp System

[0424] These new DNA constructs make use of the site specificrecombination system of the P1 phage. The P1 phage possesses arecombinase called Cre which interacts specifically with a 34 base pairsloxP site. The loxP site is composed of two palindromic sequences of 13bp separated by a 8 bp conserved sequence (Hoess et al, 1986). Therecombination by the Cre enzyme between two loxP sites having anidentical orientation leads to the deletion of the DNA fragment.

[0425] The Cre-loxP system used in combination with a homologousrecombination technique has been first described by Gu et al. (1993,1994) Briefly, a nucleotide sequence of interest to be inserted in atargeted location of the genome harbors at least two loxP sites in thesame orientation and located at the respective ends of a nucleotidesequence to be excised from the recombinant genome. The excision eventrequires the presence of the recombinase (Cre) enzyme within the nucleusof the recombinant cell host The recombinase enzyme may be brought atthe desired time either by (a) incubating the recombinant cell hosts ina culture medium containing this enzyme, by injecting the Cre enzymedirectly into the desired cell, such as described by Araki et al.(1995), or by lipofection of the enzyme into the cells, such asdescribed by Baubonis et al. (1993), (b) transfecting the cell host witha vector comprising the Cre coding sequence operably linked to apromoter functional in the recombinant cell host, which promoter beingoptionally inducible, said vector being introduced in the recombinantcell host, such as described by Gu et al (1993) and Sauer et al. (1988);(c) introducing in the genome of the cell host a polynucleotidecomprising the Cre coding sequence operably linked to a promoterfunctional in the recombinant cell host, which promoter is optionallyinducible, and said polynucleotide being inserted in the genome of thecell host either by a random insertion event or an homologousrecombination event, such as described by Gu et al (1994) In thespecific embodiment wherein the vector containing the sequence to beinserted in the RBP-7 gene by homologous recombination is constructed insuch a way that selectable markers are flanked by loxP sites of the sameorientation, it is possible, by treatment by the Cre enzyme, toeliminate the selectable markers while leaving the RBP-7 sequences ofinterest that have been inserted by an homologous recombination event.Again, two selectable markers are needed: a positive selection marker toselect for the recombination event and a negative selection marker toselect for the homologous recombination event. Vectors and methods usingthe Cre-loxP system are described by Zou et al. (1994).

[0426] Thus, a third preferred DNA construct of the invention comprises,from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprisedin the RBP-7 genomic sequence; (b) a nucleotide sequence comprising apolynucleotide encoding a positive selection marker, said nucleotidesequence comprising additionally two sequences defining a siterecognized by a recombinase, such as a loxP site, the two sites beingplaced in the same orientation; and (c) a second nucleotide sequencethat is comprised in the RBP-7 genomic sequence, and is located on thegenome downstream of the first RBP-7 nucleotide sequence (a).

[0427] The sequences defining a site recognized by a recombinase, suchas a loxP site, are preferably located within the nucleotide sequence(b) at suitable locations bordering the nucleotide sequence for whichthe conditional excision is sought. In one specific embodiment, two loxPsites are located at each side of the positive selection markersequence, in order to allow its excision at a desired time after theoccurrence of the homologous recombination event

[0428] In a preferred embodiment of a method using the third DNAconstruct described above, the excision of the polynucleotide fragmentbordered by the two sites recognized by a recombinase, preferably twoloxP sites, is performed at a desired time, due to the presence withinthe genome of the recombinant cell host of a sequence encoding the Creenzyme operably linked to a promoter sequence, preferably an induciblepromoter, more preferably a tissue-specific promoter sequence and mostpreferably a promoter sequence which is both inducible andtissue-specific, such as described by Gu et al (1994)

[0429] The presence of the Cre enzyme within the genome of therecombinant cell host may result of the breeding of two transgenicanimals, the first transgenic animal bearing the RBP-7-derived sequenceof interest containing the loxP sites as described above and the secondtransgenic animal bearing the Cre coding sequence operably linked to asuitable promoter sequence, such as described by Gu et al (1994).

[0430] Spatio-temporal control of the Cre enzyme expression may also beachieved with an adenovirus based vector that contains the Cre gene thusallowing infection of cells, or in vivo infection of organs, fordelivery of the Cre enzyme, such as described by Anton and Graham (1995)and Kanegae et al (1995).

[0431] The DNA constructs described above may be used to introduce adesired nucleotide sequence of the invention, preferably a RBP-7 genomicsequence or a RBP-7 cDNA sequence, and most preferably an altered copyof a RBP-7 genomic or cDNA sequence, within a predetermined location ofthe targeted genome, leading either to the generation of an altered copyof a targeted gene (knock-out homologous recombination) or to thereplacement of a copy of the targeted gene by another copy sufficientlyhomologous to allow an homologous recombination event to occur (knock-inhomologous recombination).

[0432] Nuclear Antisense DNA Constructs

[0433] Preferably, the antisense polynucleotides of the invention have a3′ polyadenylation signal that has been replaced with a self-cleavingribozyme sequence, such that RNA polymerase II transcripts are producedwithout poly(A) at their 3′ ends, these antisense polynucleotides beingincapable of export from the nucleus, such as described by Liu et al.(1994) In a preferred embodiment, these RBP-7 antisense polynucleotidesalso comprise, within the ribozyme cassette, a histone stem-loopstructure to stabilize cleaved transcripts against 3′-5′ exonucleolyticdegradation, such as described by Eckner et al (1991).

Cell Hosts

[0434] Another aspect of the invention is a host cell that has beentransformed or transfected with one of the polynucleotides describedherein, and in particular a polynucleotide either comprising a RBP-7regulatory polynucleotide or the coding sequence of the RBP-7polypeptide selected from the group consisting of SEQ ID Nos 1 and 4 ora fragment or a variant thereof. Also included are host cells that aretransformed (prokaryotic cells) or that are transfected (eukaryoticcells) with a recombinant vector such as one of those described above.More particularly, the cell hosts of the present invention can compriseany of the polynucleotides described in the “RBP-7 Gene, CorrespondingcDNAs And RBP-7 Coding And Regulating Sequences” section, and the“Oligonucleotide Probes And Primers” section.

[0435] A further recombinant cell host according to the inventioncomprises a polynucleotide containing a biallelic marker selected fromthe group consisting of A1 to A21, and the complements thereof.

[0436] An additional recombinant cell host according to the inventioncomprises any of the vectors described herein, more particularly any ofthe vectors described in the “Vectors For The Expression Of A RegulatoryOr A Coding Polynucleotide According To The Invention” section

[0437] All the above-described vectors are useful to transform ortransfect cell hosts in order to express a polynucleotide coding for aRBP-7 polypeptide or their peptide fragments or variants, or apolynucleotide of interest derived from the RBP-7 gene

[0438] Suitable prokaryotic hosts for transformation include E coli,Bacillus subtlis, as well as various species within the genera ofStreptomyces or Mycobacterium Suitable eukaryotic hosts comprise yeast,insect cells, such as Drosophila and Sf9. Various mammalian cell hostscan also be employed to express recombinant protein. Examples ofmammalian cell hosts include the COS-7 lines of monkey kidneyfibroblasts (Guzman, 1981), and other cell lines capable of expressing acompatible vector, for example the C 127, 3T3, CHO, HeLa and BHK celllines. The selection of an host is within the scope of the one skilledin the art.

[0439] A cell host according to the present invention is characterizedin that its genome or genetic background (including chromosome,plasmids) is modified by the heterologous nucleic acid coding for aRBP-7 polypeptide or a peptide fragment or van ant, or by apolynucleotide of interest derived from the RBP-7 gene

[0440] Preferred cell hosts used as recipients for the expressionvectors of the invention are the followings:

[0441] a) Prokaryotic cells: Escherichia coli strains (I.E. DH5-□strain) or Bacillus subtilis.

[0442] b) Eukaryotic cell hosts: HeLa cells (ATCC No. CCL2, No. CCL2.1;No. CCL2.2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL1650;No. CRL1651), Sf-9 cells (ATCC No. CRL1711), mammal ES stem cells

[0443] Preferably, the mammal ES stem cells include human (Thomson etal, 1998), mice, rats and rabbits ES stem cells and are preferably usedin a process for producing transgenic animals, such as those describedbelow.

[0444] The RBP-7 gene expression in human cells may be rendereddefective, or alternatively it may be proceeded with the insertion of aRBP-7 genomic or cDNA sequence with the replacement of the RBP-7 genecounterpart in the genome of an animal cell by a RBP-7 polynucleotideaccording to the invention. These genetic alterations may be generatedby homologous recombination events using specific DNA constructs thathave been previously described.

[0445] One kind of cell hosts that may be used are mammal zygotes, suchas murine zygotes. For example, murine zygotes may undergomicroinjection with a purified DNA molecule of interest, for example apurified DNA molecule that has previously been adjusted to aconcentration range from 1 ng/ml—for BAC inserts-3 ng/μl—for P1bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing100 mM NaCl, 30 μM spermine, and 70 μM spermidine When the DNA to bemicroinjected has a large size, polyamines and high salt concentrationscan be used in order to avoid mechanical breakage of this DNA, asdescribed by Schedl et al (1993b)

[0446] Anyone of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripotent, uncommited cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the followingES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No. CRL-11632), YS001 (ATCC No. CRL-11776), 36.5 (ATCC No. CRL-11116). Tomaintain ES cells in an uncommitted state, they are cultured in thepresence of growth inhibited feeder cells which provide the appropriatesignals to preserve thus embryonic phenotype and serve as a matrix forES cell adherence. Preferred feeder cells are primary embryonicfibroblasts that are established from tissue of day 13-day 14 embryos ofvirtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al (1993) and are inhibited in growth byirradiation, such as described by Robertson (1987), or by the presenceof an inhibitory concentration of LIF, such as described by Pease andWilliams (1990).

[0447] The constructs in the host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.

[0448] Following transformation of a suitable host and growth of thehost to an appropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

[0449] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification

[0450] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents Suchmethods are well known by the skill artisan.

[0451] Transgenic Animals

[0452] The terms “transgenic animals” or “host animals” are used hereindesignate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. Preferred animals are non-human mammals and include thosebelonging to a genus selected from Mus (e g mice), Rattus (e.g. rats)and Oryctogalus (e.g. rabbits) which have their genome artificially andgenetically altered by the insertion of a nucleic acid according to theinvention.

[0453] The transgenic animals of the invention all include within aplurality of their cells a cloned recombinant or synthetic DNA sequence,more specifically one of the purified or isolated nucleic acidscomprising a RBP-7 coding sequence, a RBP-7 regulatory polynucleotide ora DNA sequence encoding an antisense polynucleotide such as described inthe present specification.

[0454] Preferred transgenic animals according to the invention containsin their somatic cells and/or in their germ line cells any one of thepolynucleotides, the recombinant vectors and the cell hosts described inthe present invention. More particularly, the transgenic animals of thepresent invention can comprise any of the polynucleotides described inthe “RBP-7 Gene, Corresponding cDNAs And RBP-7 Coding And RegulatingSequences” section, the “Oligonucleotide Probes And Primers” section,the “Vectors For The Expression Of A Regulatory Or A CodingPolynucleotide According To The Invention” section and the “Cell Hosts”section.

[0455] The transgenic animals of the invention thus contain specificsequences of exogenous genetic material such as the nucleotide sequencesdescribed above in detail.

[0456] In a first preferred embodiment, these transgenic animals may begood experimental models in order to study the diverse pathologiesrelated to cell differentiation, in particular concerning the transgenicanimals within the genome of which has been inserted one or severalcopies of a polynucleotide encoding a native RBP-7 protein, oralternatively a mutant RBP-7 protein.

[0457] In a second preferred embodiment, these transgenic animals mayexpress a desired polypeptide of interest under the control of theregulatory polynucleotides of the RBP-7 gene, leading to good yields inthe synthesis of this protein of interest, and eventually a tissuespecific expression of this protein of interest

[0458] The design of the transgenic animals of the invention may be madeaccording to the conventional techniques well known from the one skilledin the art. For more details regarding the production of transgenicanimals, and specifically transgenic mice, it may be referred to Sandouet al. (1994) and also to U.S. Pat. No. 4,873,191, issued Oct. 10, 1989,U.S. Pat. No. 5,464,764 issued Nov. 7, 1995 and U.S. Pat. No. 5,789,215,issued Aug 4, 1998,these documents being herein incorporated byreference to disclose methods producing transgenic mice.

[0459] Transgenic animals of the present invention are produced by theapplication of procedures which result in an animal with a genome thathas incorporated exogenous genetic material. The procedure involvesobtaining the genetic material, or a portion thereof, which encodeseither a RBP-7 coding sequence, a RBP-7 regulatory polynucleotide or aDNA sequence encoding a RBP-7 antisense polynucleotide such as describedin the present specification.

[0460] A recombinant polynucleotide of the invention is inserted into anembryonic or ES stem cell line. The insertion is preferably made usingelectroporation, such as described by Thomas et al. (1987) The cellssubjected to electroporation are screened (e.g. by selection viaselectable markers, by PCR or by Southern blot analysis) to findpositive cells which have integrated the exogenous recombinantpolynucleotide into their genome, preferably via an homologousrecombination event. An illustrative positive-negative selectionprocedure that may be used according to the invention is described byMansour et al. (1988)

[0461] Then, the positive cells are isolated, cloned and injected into3.5 days old blastocysts from mice, such as described by Bradley (1987).The blastocysts are then inserted into a female host animal and allowedto grow to term.

[0462] Alternatively, the positive ES cells are brought into contactwith embryos at the 2.5 days old 8-16 cell stage (morulae) such asdescribed by Wood et al. (1993) or by Nagy et al. (1993), the ES cellsbeing internalized to colonize extensively the blastocyst including thecells which will give rise to the germ line

[0463] The offsprings of the female host are tested to determine whichanimals are transgenic e.g. include the inserted exogenous DNA sequenceand which are wild-type.

[0464] Thus, the present invention also concerns a transgenic animalcontaining a nucleic acid, a recombinant expression vector or arecombinant host cell according to the invention.

[0465] Recombinant Cell Lines Derived from the Transgenic Animals of theInvention

[0466] A further aspect of the invention is recombinant cell hostsobtained from a transgenic animal described herein.

[0467] Recombinant cell lines may be established in vitro from cellsobtained from any tissue of a transgenic animal according to theinvention, for example by transfection of primary cell cultures withvectors expressing onc-genes such as SV40 large T antigen, as describedby Chou (1989) and Shay et al (1991).

RBP-7 Polypeptides

[0468] It is now easy to produce proteins in high amounts by geneticengineering techniques through expression vectors such as plasmids,phages or phagemids. The polynucleotide that code for one thepolypeptides of the present invention is inserted in an appropriateexpression vector in order to produce in vitro the polypeptide ofinterest

[0469] Thus, the present invention also concerns a method for producingone of the polypeptides described herein, and especially a polypeptideof SEQ ID No. 29 or a fragment or a variant thereof, wherein said methodcomprises the steps of:

[0470] a) Optionally amplifying the nucleic acid coding for a RBP-7polypeptide, or a fragment or a variant thereof, using a pair of primersaccording to the invention (by PCR, SDA, TAS, 3SR NASBA, TMA etc.)

[0471] b) Inserting the resulting amplified nucleic acid in anappropriate vector;

[0472] c) culturing, in an appropriate culture medium, a cell hostpreviously transformed or transfected with the recombinant vector ofstep b);

[0473] d) harvesting the culture medium thus conditioned or lyse thecell host, for example by sonication or by an osmotic shock;

[0474] e) separating or purifying, from the said culture medium, or fromthe pellet of the resultant host cell lysate the thus producedpolypeptide of interest.

[0475] f) Optionally characterizing the produced polypeptide ofinterest.

[0476] The polypeptides according to the invention may be characterizedby binding onto an immunoaffinity chromatography column on whichpolyclonal or monoclonal antibodies directed to a polypeptide of SEQ IDNo. 29, or a fragment or a van ant thereof, have previously beenimmobilized

[0477] Purification of the recombinant proteins or peptides according tothe present invention may be carried out by passage onto a Nickel orCupper affinity chromatography column. The Nickel chromatography columnmay contain the Ni-NTA resin (Porath et al., 1975).

[0478] The polypeptides or peptides thus obtained may be purified, forexample by high performance liquid chromatography, such as reverse phaseand/or cationic exchange HPLC, as described by Rougeot et al. (1994).The reason to prefer this kind of peptide or protein purification is thelack of byproducts found in the elution samples which renders theresultant purified protein or peptide more suitable for a therapeuticuse.

[0479] Another aspect of the present invention comprises a purified orisolated RBP-7 polypeptide or a fragment or a variant thereof

[0480] In a preferred embodiment, the RBP-7 polypeptide comprises anamino acid sequence of SEQ ID No. 29 or a fragment or a variant thereofIn a further embodiment, the present invention embodies isolated,purified, and recombinant polypeptides comprising a contiguous span ofat least 6 amino acids, preferably at least 8 to 10 amino acids, morepreferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids ofSEQ ID No. 29.

[0481] The RBP-7 polypeptide of the amino acid sequence of SEQ ID No. 29has 1312 amino acids in length This 1312 amino acid sequence harborsnotably potential sites indicating post-translational modifications suchas 8 N-glycosylation sites, 72 phosphorylation sites, 8 N-myristoylationsites and 4 amidation sites. The location of these sites is referred toin the appended Sequence Listing when disclosing the features of theamino acid sequence of SEQ ID No 29

[0482] The RBP-7 polypeptide shares some homology in amino acid sequencewith another retinoblastoma binding protein, namely human RBP-1 (Fattaeyet al, 1993). More precisely, a 48% identity has been found betweenRBP-7 and RBP-1 for the amino acid sequence beginning at position 1 andending at position 790 of RBP-7. A 30% identity has been found for theamino acid sequence beginning at position 791 and ending at position1312 of RBP-7.

[0483] A further object of the present invention concerns a purified orisolated polypeptide which is encoded by a nucleic acid comprising anucleotide sequence selected from the group consisting of SEQ ID Nos 1,4 and 5-28 or fragments or variants thereof Preferably, the purified orisolated polypeptide comprises at least 10, at least 15, at least 20 orat least 25 consecutive amino acids of the polypeptides encoded by SEQID Nos 1, 4 and 5-28.

[0484] The invention includes a nucleic acid encoding a RBP-7polypeptide comprising at least one of the biallelic markers of thepresent invention, more particularly at least one of the biallelicmarkers defined in SEQ ID No. 30-71.

[0485] More generally, the invention also pertains to a variant RBP-7polypeptide comprising at least one amino acid substitution, addition ordeletion, when compared with the sequence of SEQ ID No. 29. Moreparticularly, the invention encompasses a RBP-7 protein or a fragmentthereof comprising a contiguous span of at least 6 amino acids,preferably at least 8 to 10 amino acids, more preferably at least 12,15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 29 comprisingat least one of the following amino acids:

[0486] a Glycine residue at the ammo acid position 293 of SEQ ID No 29;

[0487] a Glutamic acid at the amino acid in position 963 of SEQ ID No.29;

[0488] a Methionine residue at the amino acid position 969 of SEQ ID No29.

[0489] A variant or mutated RBP-7 polypeptide comprises amino acidchanges of at least one amino acid substitution, deletion or addition,preferably from 1 to 10, 20 or 30 amino acid substitutions or additionsThe ammo acid substitutions are generally non conservative in terms ofpolarity, charge, hydrophilicity properties of the substitute amino acidwhen compared with the native amino acid. The amino acid changesoccurring in such a mutated RBP-7 polypeptide may be determinant for thebiological activity or for the capacity of the mutated RBP-7 polypeptideto be recognized by antibodies raised against a native RBP-7

[0490] Such a variant or mutated RBP-7 protein may be the target ofdiagnostic tools, such as specific monoclonal or polyclonal antibodies,useful for detecting the mutated RBP-7 protein in a sample

[0491] Are also part of the present invention polypeptides that arehomologous to a RBP-7 polypeptide, especially a polypeptide of SEQ IDNo. 29, or their fragments or variants.

[0492] The invention also encompasses a RBP-7 polypeptide or a fragmentor a variant thereof in which at least one peptide bound has beenmodified as described in “Definitions”.

[0493] The polypeptides according to the invention may also be preparedby the conventional methods of chemical synthesis, either in ahomogenous solution or in solid phase. As an illustrative embodiment ofsuch chemical polypeptide synthesis techniques, it may be cited thehomogenous solution technique described by Houbenweyl in 1974.

[0494] The RBP-7 polypeptide, or a fragment or a variant thereof maythus be prepared by chemical synthesis in liquid or solid phase bysuccessive couplings of the different ammo acid residues to beincorporated (from the N-terminal end to the C-terminal end in liquidphase, or from the C-terminal end to the N-terminal end in solid phase)wherein the N-terminal ends and the reactive side chains are previouslyblocked by conventional groups

[0495] For solid phase synthesis the technique described by Merrifield(1965) may be used in particular.

Antibodies

[0496] The polypeptides according to the present invention, especiallythe polypeptides of SEQ ID No. 29 are allowing the preparation ofpolyclonal or monoclonal antibodies that recognize the polypeptides ofSEQ ID No. 29 or fragments thereof

[0497] The antibodies may be prepared from hybridomas according to thetechnique described by Kohler and Milstein in 1975. The polyclonalantibodies may be prepared by immunization of a mammal, especially amouse or a rabbit, with a polypeptide according to the invention that iscombined with an adjuvant of immunity, and then by purifying of thespecific antibodies contained in the serum of the immunized animal on aaffinity chromatography column on which has previously been immobilizedthe polypeptide that has been used as the antigen

[0498] The invention also concerns a purified or isolated antibodycapable of specifically binding to the RBP-7 protein, more particularlyto selected peptide fragments thereof, and more preferably polypeptidesencoded by nucleic acids comprising one or more biallelic markers of theinvention, or a van ant thereof. In addition, the invention comprisesantibodies capable of specifically binding to a fragment or variant ofsuch a RBP-7 protein comprising an epitope of the RBP-7 protein,preferably an antibody capable of binding to a polypeptide comprising atleast 10 consecutive amino acids, at least 15 consecutive amino acids,at least 20 consecutive amino acids, or at least 40 consecutive aminoacids of a RBP-7 protein, more preferably an antibody capable of bindingspecifically to a variant or mutated RBP-7 protein or a fragment thereofand distinguishing between either two variants of RBP-7 or mutated RBP-7and non-mutated RBP-7 protein.

[0499] The proteins expressed from a RBP-7 DNA comprising at least oneof the nucleic sequences of SEQ ID Nos 30-71 or a fragment or a variantthereof, preferably the nucleic sequences of the biallelic markersleading to an amino acid substitution, may also be used to generateantibodies capable of specifically binding to the expressed RBP-7protein or fragments or variants thereof.

[0500] In another embodiment, polyclonal or monoclonal antibodiesaccording to the invention are raised against a RBP-7 polypeptidecomprising at least one of the following amino acids.

[0501] a Glycine residue at the amino acid position 293 of SEQ ID No 29;

[0502] a Glutamic acid at the amino acid in position 963 of SEQ ID No29;

[0503] a Methionine residue at the amino acid position 969 of SEQ ID No.29.

[0504] Alternatively, the antibodies may be capable of binding fragmentsof the RBP-7 protein which comprise at least 10 amino acids encoded bythe sequences of SEQ ID Nos 1 and 4, preferably comprising at least oneof the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereofIn some embodiments, the antibodies may be capable of binding fragmentsof the RBP-7 protein which comprise at least 15 ammo acids encoded bythe sequences of SEQ ID Nos 1 and 4, preferably comprising at least oneof the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereof.In other embodiments, the antibodies may be capable of binding fragmentsof the RBP-7 protein which comprise at least 25 amino acids encoded bythe sequences of SEQ ID Nos 1 and 4, preferably comprising at least oneof the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereofin further embodiments, the antibodies may be capable of bindingfragments of the RBP-7 protein which comprise at least 40 amino acidsencoded by the sequences of SEQ ID Nos 1 and 4, preferably comprising atleast one of the sequences of SEQ ID Nos 30-71 or a fragment or avariant thereof

[0505] Both monoclonal antibodies and polyclonal antibodies are withinthe scope of the present invention Monoclonal or polyclonal antibodiesto the protein can then be prepared as follows:

[0506] A. Monoclonal Antibody Production by Hybridoma Fusion

[0507] Monoclonal antibody to epitopes in the RBP-7 protein or a portionthereof can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein, (1975) or derivative methodsthereof Briefly, a mouse is repetitively inoculated with a fewmicrograms of the RBP-7 protein or a portion thereof over a period of afew weeks. The mouse is then sacrificed, and the antibody producingcells of the spleen isolated The spleen cells are fused by means ofpolyethylene glycol with mouse myeloma cells, and the excess unfusedcells destroyed by growth of the system on selective media comprisingaminopterin (HAT media). The successfully fused cells are diluted andaliquots of the dilution placed in wells of a microtiter plate wheregrowth of the culture is continued Antibody-producing clones areidentified by detection of antibody in the supernatant fluid of thewells by immunoassay procedures, such as ELISA, as originally describedby Engvall, (1980), and derivative methods thereof Selected positiveclones can be expanded and their monoclonal antibody product harvestedfor use. Detailed procedures for monoclonal antibody production aredescribed in Davis, L. et al

[0508] B. Polyclonal Antibody Production by Immunization

[0509] Polyclonal antiserum containing antibodies to heterogeneousepitopes in the RBP-7 protein or a portion thereof can be prepared byimmunizing suitable animals with the RBP-7 protein or a portion thereof,which can be unmodified or modified to enhance immunogenicity. Effectivepolyclonal antibody production is affected by many factors related bothto the antigen and the host species. For example, small molecules tendto be less immunogenic than others and may require the use of carriersand adjuvant. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera Small doses (ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, (1971).

[0510] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al. (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum. Affinityof the antisera for the antigen is determined by preparing competitivebinding curves, as described, for example, by Fisher (1980).

[0511] Antibody preparations prepared according to either protocol areuseful in quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for lulling cells expressing the protein or reducing thelevels of the protein in the body.

[0512] Consequently, the invention is also directed to a method fordetecting specifically the presence of a polypeptide according to theinvention in a biological sample, said method comprising the followingsteps

[0513] a) bringing into contact the biological sample with an antibodyaccording to the invention,

[0514] b) detecting the antigen-antibody complex formed

[0515] Another aspect of the invention is a diagnostic kit for in vitrodetecting the presence of a polypeptide according to the presentinvention in a biological sample, wherein said kit comprises

[0516] a) a polyclonal or monoclonal antibody as described above,optionally labeled;

[0517] b) a reagent allowing the detection of the antigen-antibodycomplexes formed, said reagent carrying optionally a label, or beingable to be recognized itself by a labeled reagent, more particularly inthe case when the above-mentioned monoclonal or polyclonal antibody isnot labeled by itself

Methods for Screening Substances Interacting with a RBP-7 Polypeptide

[0518] For the purpose of the present invention, a ligand means amolecule, such as a protein, a peptide, an antibody or any syntheticchemical compound capable of binding to the RBP-7 protein or one of itsfragments or variants or to modulate the expression of thepolynucleotide coding for RBP-7 or a fragment or variant thereof

[0519] In the ligand screening method according to the presentinvention, a biological sample or a defined molecule to be tested as aputative ligand of the RBP-7 protein is brought into contact with thepurified RBP-7 protein, for example the purified recombinant RBP-7protein produced by a recombinant cell host as described hereinbefore,in order to form a complex between the RBP-7 protein and the putativeligand molecule to be tested.

[0520] The present invention pertains to methods for screeningsubstances of interest that interact with a RBP-7 protein or onefragment or variant thereof By their capacity to bind covalently ornon-covalently to a RBP-7 protein or to a fragment or variant thereof,these substances or molecules may be advantageously used both in vitroand in vivo.

[0521] In vitro, said interacting molecules may be used as detectionmeans in order to identify the presence of a RBP-7 protein in a sample,preferably a biological sample

[0522] A method for the screening of a candidate substance comprises thefollowing steps

[0523] a) providing a polypeptide comprising, consisting essentially of,or consisting of a RBP-7 protein or a fragment or a variant thereof;

[0524] b) obtaining a candidate substance;

[0525] c) bringing into contact said polypeptide with said candidatesubstance;

[0526] d) detecting the complexes formed between said polypeptide andsaid candidate substance

[0527] In one embodiment of the screening method defined above, thecomplexes formed between the polypeptide and the candidate substance arefurther incubated in the presence of a polyclonal or a monoclonalantibody that specifically binds to the RBP-7 protein or to saidfragment or variant thereof

[0528] Various candidate substances or molecules can be assayed forinteraction with a RBP-7 polypeptide. These substances or moleculesinclude, without being limited to, natural or synthetic organiccompounds or molecules of biological origin such as polypeptides. Whenthe candidate substance or molecule comprises a polypeptide, thispolypeptide may be the resulting expression product of a phage clonebelonging to a phage-based random peptide library, or alternatively thepolypeptide may be the resulting expression product of a cDNA librarycloned in a vector suitable for performing a two-hybrid screening assay.

[0529] The invention also pertains to kits useful for performing thehereinbefore described screening method Preferably, such kits comprise aRBP-7 polypeptide or a fragment or a variant thereof, and optionallymeans useful to detect the complex formed between the RBP-7 polypeptideor its fragment or variant and the candidate substance In a preferredembodiment the detection means are monoclonal or polyclonal antibodiesdirected against the RBP-7 polypeptide or a fragment or a variantthereof

[0530] A. Candidate Ligands Obtained form Random Peptide Libraries

[0531] In a particular embodiment of the screening method, the putativeligand is the expression product of a DNA insert contained in a phagevector (Parmley and Smith, 1988). Specifically, random peptide phageslibraries are used The random DNA inserts encode for peptides of 8 to 20amino acids in length (Oldenburg K. R. et al., 1992.; Valadon P., etal., 1996.; Lucas A.H., 1994; Westerink M. A. J., 1995; Castagnoli L. etal. (Felici F.), 1991). According to this particular embodiment, therecombinant phages expressing a protein that binds to the immobilizedRBP-7 protein is retained and the complex formed between the RBP-7protein and the recombinant phage may be subsequently immunoprecipitatedby a polyclonal or a monoclonal antibody directed against the RBP-7protein.

[0532] Once the ligand library in recombinant phages has beenconstructed, the phage population is brought into contact with theimmobilized RBP-7 protein. Then the preparation of complexes is washedin order to remove the non-specifically bound recombinant phages. Thephages that bind specifically to the RBP-7 protein are then eluted by abuffer (acid pH) or immunoprecipitated by the monoclonal antibodyproduced by the hybridoma anti-RBP-7, and this phage population issubsequently amplified by an over-infection of bacteria (for example E.coli). The selection step may be repeated several times, preferably 2-4times, in order to select the more specific recombinant phage clones.The last step involves characterizing the peptide produced by theselected recombinant phage clones either by expression in infectedbacteria and isolation, expressing the phage insert in anotherhost-vector system, or sequencing the insert contained in the selectedrecombinant phages

[0533] B. Candidate Ligands Obtained Through a Two-Hybrid ScreeningAssay

[0534] The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989), and relies upon the fusionof a bait protein to the DNA binding domain of the yeast Gal4 protein.This technique is also described in the U.S. Pat. No. 5,667,973 and theU.S. Pat. No. 5,283,173 (Fields et al.) the technical teachings of bothpatents being herein incorporated by reference

[0535] The general procedure of library screening by the two-hybridassay may be performed as described by Harper et al. (Harper J W et al.,1993) or as described by Cho et al. (1998) or also Fromont-Racine et al(1997).

[0536] The bait protein or polypeptide comprises, consists essentiallyof, or consists of a RBP-7 polypeptide or a fragment or variant thereof

[0537] More precisely, the nucleotide sequence encoding the RBP-7polypeptide or a fragment or variant thereof is fused to apolynucleotide encoding the DNA binding domain of the GALA protein, thefused nucleotide sequence being inserted in a suitable expressionvector, for example pAS2 or pM3.

[0538] Then, a human cDNA library is constructed in a specially designedvector, such that the human cDNA insert is fused to a nucleotidesequence in the vector that encodes the transcriptional domain of theGAL4 protein Preferably, the vector used is the pACT vector. Thepolypeptides encoded by the nucleotide inserts of the human cDNA libraryare termed “pray” polypeptides.

[0539] A third vector contains a detectable marker gene, such as βgalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pGSEC may be used

[0540] Two different yeast strains are also used As an illustrative butnon limiting example the two different yeast strains may be thefollowings

[0541] Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12,trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3,cyh′);

[0542] Y187, the phenotype of which is (MATa gal4gal80 his3 trp1-901ade2-101 ura3-52 leu2-3, −112 URA3 GAL-lacZmef), which is the oppositemating type of Y190.

[0543] Briefly, 20 μg of pAS2/RBP-7 and 20 μg of pACT-cDNA library areco-transformed into yeast strain Y190. The transformants are selectedfor growth on minimal media lacking histidine, leucine and tryptophan,but containing the histidine stnthesis inhibitor 3-AT (50 mM). Positivecolonies are screened for beta galactosidase by filter lift assay. Thedouble positive colonies (His⁺, β-gal⁺) are then grown on plates lackinghistidine, leucine, but containing tryptophan and cycloheximide (10mg/ml) to select for loss of pAS2/RBP-7 plasmids bu retention ofpACT-cDNA library plasmids. The resulting Y190 strains are mated withY187 strains expressing RBP-7 or non-related control proteins; such ascyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper etal (Harper J W et al., 1993) and by Bram et al (Bram R J et al, 1993),and screened for β galactosidase by filter lift assay Yeast clones thatare β gal—after mating with the control Gal4 fusions are consideredfalse positives

[0544] In another embodiment of the two-hybrid method according to theinvention, interaction between RBP-7 or a fragment or variant thereofwith cellular proteins may be assessed using the Matchmaker Two HybridSystem 2 (Catalog No K 1604-1, Clontech) As described in the manualaccompanying the Matchmaker Two Hybrid System 2 (Catalog No K1604-1,Clontech), the disclosure of which is incorporated herein by reference,nucleic acids encoding the RBP-7 protein or a portion thereof, areinserted into an expression vector such that they are in frame with DNAencoding the DNA binding domain of the yeast transcriptional activatorGAL4. A desired cDNA, preferably human cDNA, is inserted into a secondexpression vector such that they are in frame with DNA encoding theactivation domain of GAL4. The two expression plasmids are transformedinto yeast and the yeast are plated on selection medium which selectsfor expression of selectable markers on each of the expression vectorsas well as GAL4 dependent expression of the HIS3 gene. Transformantscapable of growing on medium lacking histidine are screened for GAL4dependent lacZ expression. Those cells which are positive in both thehistidine selection and the lacZ assay contain interaction between RBP-7and the protein or peptide encoded by the initially selected cDNAinsert.

[0545] C. Candidate Ligand Obtained Through Biosensor Assay

[0546] Proteins interacting with the RBP-7 protein or portions thereofcan also be screened by using an Optical Biosensor as described inEdwards et Leatherbarrow (1997), the disclosure of which is incorporatedherein by reference. The main advantage of the method is that it allowsthe determination of the association rate between the protein and otherinteracting molecules. Thus, it is possible to specifically selectinteracting molecules with a high or low association rate. Typically atarget molecule is linked to the sensor surface (through a carboxymethldextran matrix) and a sample of test molecules is placed in contact withthe target molecules. The binding of a test molecule to the targetmolecule causes a change in the refractive index and/or thickness Thischange is detected by the Biosensor provided it occurs in the evanescentfield (which extend a few hundred nanometers from the sensor surface) Inthese screening assays, the target molecule can be the RBP-7 protein ora portion thereof and the test sample can be a collection of proteinsextracted from tissues or cells, a pool of expressed proteins,combinatorial peptide and/or chemical libraries, or phage displayedpeptides. The tissues or cells from which the test proteins areextracted can originate from any species.

[0547] Method for Screening Ligands that Modulate the Expression of theRBP-7 Gene

[0548] The present invention also concerns a method for screeningsubstances or molecules that are able to increase, or in contrast todecrease, the level of expression of the RBP-7 gene. Such a method mayallow the one skilled in the art to select substances exerting aregulating effect on the expression level of the RBP-7 gene and whichmay be useful as active ingredients included in pharmaceuticalcompositions for treating patients suffering from deficiencies in theregulation of expression of the RBP-7 gene

[0549] Thus, another aspect of the present invention is a method for thescreening of a candidate substance or molecule, said method comprisingthe following steps:

[0550] a) providing a recombinant cell host containing a nucleic acid,wherein said nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID Nos. 1, 4, 30-75 or the sequencescomplementary thereto or a fragment or a variant thereof;

[0551] b) obtaining a candidate substance, and

[0552] c) determining the ability of the candidate substance to modulatethe expression levels of the nucleic acid comprising a nucleotidesequence selected from the group consisting of SEQ ID No: 1, 4, 30-75 orthe sequences complementary thereto or a fragment or a variant thereof.

[0553] The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea recombinant vector that allows the expression of a nucleic acidcomprising a nucleotide sequence selected from the group consisting ofSEQ ID No: 1, 4, 30-75 or the sequences complementary thereto or afragment or a variant thereof or, alternatively, the kit may comprise arecombinant cell host containing such recombinant vectors

[0554] Another subject of the present invention is a method forscreening molecules that modulate the expression of the RBP-7 protein.Such a screening method comprises the steps of:

[0555] a) cultivating a prokaryotic or an eukaryotic cell that has beentransfected with a nucleotide sequence encoding the RBP-7 protein,placed under the control of its own promoter,

[0556] b) bringing into contact the cultivated cell with a molecule tobe tested,

[0557] c) quantifying the expression of the RBP-7 protein

[0558] In another embodiment of a method for screening of a candidatesubstance or molecule that modulates the expression of the RBP-7 gene,the method comprises the following steps:

[0559] a) providing a recombinant cell host containing a nucleic acid,wherein said nucleic acid comprises the nucleotide sequence of SEQ IDNo. 2, the sequence complementary thereto, or a biologically activefragment or variant thereof located upstream a polynucleotide encoding adetectable protein,

[0560] b) obtaining a candidate substance, and

[0561] c) determining the ability of the candidate substance to modulatethe expression levels of the polynucleotide encoding the detectableprotein.

[0562] Among the preferred polynucleotides encoding a detectableprotein, there may be cited polynucleotides encoding β galactosidase,green fluorescent protein (GFP) and chloramphenicol acetyl transferase(CAT).

[0563] The invention also pertains to kits useful for performing thehereinbefore described screening method Preferably, such kits comprise arecombinant vector that allows the expression of a nucleotide sequenceof SEQ ID No 2 or a biologically active fragment or variant thereoflocated upstream a polynucleotide encoding a detectable protein

[0564] For the design of suitable recombinant vectors useful forperforming the screening methods described above, it will be referred tothe section of the present specification wherein the preferredrecombinant vectors of the invention are detailed.

[0565] Using DNA recombination techniques well known by the one skill inthe art, the RBP-7 protein encoding DNA sequence is inserted into anexpression vector, downstream from its promoter sequence. As anillustrative example, the promoter sequence of the RBP-7 gene iscontained in the nucleic acid of SEQ ID No. 2.

[0566] The quantification of the expression of the RBP-7 protein may berealized either at the mRNA level or at the protein level In the lattercase, polyclonal or monoclonal antibodies may be used to quantify theamounts of the RBP-7 protein that have been produced, for example in anELISA or a RIA assay.

[0567] In a preferred embodiment, the quantification of the RBP-7 mRNAis realized by a quantitative PCR amplification of the cDNA obtained bya reverse transcription of the total mRNA of the cultivatedRBP-7-transfected host cell, using a pair of primers specific for RBP-7.

[0568] Expression levels and patterns of RBP-7 may be analyzed bysolution hybridization with long probes as described in InternationalPatent Application No WO 97/05277, the entire contents of which areincorporated herein by reference. Briefly, the RBP-7 cDNA or the RBP-7genomic DNA described above, or fragments thereof, is inserted at acloning site immediately downstream, of a bacteriophage (T3, T7 or SP6)RNA polymerase promoter to produce antisense RNA. Preferably, the RBP-7insert comprises at least 100 or more consecutive nucleotides of thegenomic DNA sequence or the cDNA sequences, particularly thosecomprising at least one of SEQ ID Nos 30-71 or those encoding mutatedRBP-7. The plasmid is linearized and transcribed in the presence ofribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP andDIG-UTP). An excess of this doubly labeled RNA is hybridized in solutionwith mRNA isolated from cells or tissues of interest. The hybridizationsare performed under standard stringent conditions (40-50° C. for 16hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The unhybridizedprobe is removed by digestion with ribonucleases specific forsingle-stranded RNA (i e. RNases CL3, T1, Phy M, U2 or A). The presenceof the biotin-UTP modification enables capture of the hybrid on amicrotitration plate coated with streptavidin The presence of the DIGmodification enables the hybrid to be detected and quantified by ELISAusing an anti-DIG antibody coupled to alkaline phosphatase.

Methods for Inhibiting the Expression of a RBP-7 Gene

[0569] Other therapeutic compositions according to the present inventioncomprise advantageously an oligonucleotide fragment of the nucleicsequence of RBP-7 as an antisense tool or a triple helix tool thatinhibits the expression of the corresponding BP-7 gene.

[0570] A—Antisense Approach

[0571] Preferred methods using antisense polynucleotide according to thepresent invention are the procedures described by Sczakiel et al.(Sczakiel G. et al, 1995).

[0572] Preferably, the antisense tools are choosen among thepolynucleotides (15-200 bp long) that are complementary to the 5′ en dof the RBP-7 mRNA. In another embodiment, a combination of differentantisense polynucleotides complementary to different parts of thedesired targetted gene are used.

[0573] Preferred antisense polynucleotides according to the presentinvention are complementary to a sequence of the mRNAs of RBP-7 thatcontains the translation initiation codon ATG.

[0574] The antisense nucleic acid molecules to be used in gene therapymay be either DNA or RNA sequences They comprise a nucleotide sequencecomplementary to the targeted sequence of the RBP-7 genomic DNA, thesequence of which can be determined using one of the detection methodsof the present invention. In a preferred embodiment, the antisenseoligonucleotide are able to hybridize with at least one of the splicingsites of the targeted RBP-7 gene, or with the 3′ UTR of the 5′ U TR. Theantisense nucleic acids should have a length and melting temperaturesufficient to permit formation of an intracellular duplex havingsufficient stability to inhibit the expression of the RBP-7 mRNA in theduplex Strategies for designing antisense nucleic acids suitable for usein gene therapy are disclosed in Green et al., (1986) and Izant andWeintraub, (1984), the disclosures of which are incorporated herein byreference

[0575] In some strategies, antisense molecules are obtained by reversingthe orientation of the RBP-7 coding region with respect to a promoter soas to transcribe the opposite strand from that which is normallytranscribed in the cell The antisense molecules may be transcribed usingin vitro transcription systems such as those which employ T7 or SP6polymerase to generate the transcript. Another approach involvestranscription of RBP-7 antisense nucleic acids in vivo by operablylinking DNA containing the antisense sequence to a promoter in asuitable expression vector

[0576] Alternatively, suitable antisense strategies are those describedby Rossi et al. (1991), in the International Applications Nos WO94/23026, WO 95/04141, WO 92/18522 and in the European PatentApplication No. EP 0 572 287 A2, the disclosures of which areincorporated herein by reference in their entireties.

[0577] An alternative to the antisense technology that is used accordingto the present invention involves using ribozymes that will bind to atarget sequence via their complementary polynucleotide tail and thatwill cleave the corresponding RNA by hydrolyzing its target site (namely“hammerhead ribozymes”). Briefly, the simplified cycle of a hammerheadribozyme involves (1) sequence specific binding to the target RNA viacomplementary antisense sequences; (2) site-specific hydrolysis of thecleavable motif of the target strand; and (3) release of cleavageproducts, which gives rise to another catalytic cycle. Indeed, the useof long-chain antisense polynucleotide (at least 30 bases long) orribozymes with long antisense arms are advantageous. A preferreddelivery system for antisense ribozyme is achieved by covalently linkingthese antisense ribozymes to lipophilic groups or to use liposomes as aconvenient vector. Preferred antisense ribozymes according to thepresent invention are prepared as described by Sczakiel et al. (1995),the specific preparation procedures being referred to in said articlebeing herein incorporated by reference.

[0578] B—Triple Helix Approach

[0579] The RBP-7 genomic DNA may also be used to inhibit the expressionof the RBP-7gene based on intracellular triple helix formation.

[0580] Triple helix oligonucleotides are used to inhibit transcriptionfrom a genome. They are particularly useful for studying alterations incell activity when it is associated with a particular gene.

[0581] Similarly, a portion of the RBP-7 genomic DNA can be used tostudy the effect of inhibiting RBP-7 transcription within a cell.Traditionally, homopurine sequences were considered the most useful fortriple helix strategies However, homopyrimidine sequences can alsoinhibit gene expression Such homopyrimidine oligonucleotides bind to themajor groove at homopurine homopyrimidine sequences. Thus, both types ofsequences from the RBP-7 genomic DNA are contemplated within the scopeof this invention.

[0582] To carry out gene therapy strategies using the triple helixapproach, the sequences of the RBP-7 genomic DNA are first scanned toidentify 10-mer to 20-mer homopyrimidine or homopurine stretches whichcould be used in triple-helix based strategies for inhibiting RBP-7expression. Following identification of candidate homopyrimidine orhomopurine stretches, their efficiency in inhibiting RBP-7 expression isassessed by introducing varying amounts of oligonucleotides containingthe candidate sequences into tissue culture cells which express theRBP-7 gene.

[0583] The oligonucleotides can be introduced into the cells using avariety of methods known to those skilled in the art, including but notlimited to calcium phosphate precipitation, DEAE-Dextran,electroporation, liposome-mediated transfection or native uptake

[0584] Treated cells are monitored for altered cell function or reducedRBP-7 expression using techniques such as Northern blotting, RNaseprotection assays, or PCR based strategies to monitor the transcriptionlevels of the RBP-7 gene in cells which have been treated with theoligonucleotide

[0585] The oligonucleotides which are effective in inhibiting geneexpression in tissue culture cells may then be introduced in vivo usingthe techniques described above in the antisense approach at a dosagecalculated based on the in vitro results, as described in antisenseapproach.

[0586] In some embodiments, the natural (beta) anomers of theoligonucleotide units can be replaced with alpha anomers to render theoligonucleotide more resistant to nucleases. Further, an intercalatingagent such as ethidium bromide, or the like, can be attached to the 3′end of the alpha oligonucleotide to stabilize the triple helix. Forinformation on the generation of oligonucleotides suitable for triplehelix formation see Griffin et al. (1989), which is hereby incorporatedby this reference.

[0587] Throughout this application, various references are referred towithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the sate of the art to which this inventionpertains.

EXAMPLES Example 1

[0588] Analysis of the mRNAs Encoding a RBP-7 Polypeptide Synthesized bythe Cells

[0589] RBP-7 cDNA was obtained as follows: 4 μl of ethanol suspensioncontaining 1 mg of human prostate total RNA (Clontech laboratories,Inc., Palo Alto, USA, Catalogue N 64038-1) was centrifuged, and theresulting pellet was air dried for 30 minutes at room temperature.

[0590] First strand cDNA synthesis was performed using the Advantage™RT-for-PCR kit (Clontech laboratories Inc, catalogue N. K1402-1). 1 μlof 20 mM solution of a specific oligo dT primer was added to 12.5 μl ofRNA solution in water, heated at 74° C. for 2.5 min and rapidly quenchedin an ice bath. 10 μl of 5×RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl,3 mM MgCl₂), 2 5 μl of dNTP mix (10 mM each), 1.25 μl of humanrecombinant placental RNA inhibitor were mixed with 1 ml of MMLV reversetranscriptase (200 units). 6 5 μl of this solution were added toRNA-primer mix and incubated at 42° C. for one hour 80 μl of water wereadded and the solution was incubated at 94° C. for 5 minutes

[0591] 5 μl of the resulting solution were used in a Long Range PCRreaction with hot start, in 50 μl final volume, using 2 units of rtTHXL,20 pmol/μl of each of 5′-CCCTTGATGAGCCTCCCTATTTGACAG-3′ (SEQ ID No. 137)and 5′-CGCATTGAAATTCCCACGTCGTATTGCCAG-3′ (SEQ ID No. 138) primers with35 cycles of elongation for 6 minutes at 67° C. in thermocycler

[0592] The amplification products corresponding to both cDNA strands arepartially sequenced in order to ensure the specificity of theamplification reaction

[0593] Results of Northern blot analysis of prostate mRNAs support theexistence of a major RBP-7 cDNA having about 6 kb in length, which isapproximately the size of the longest possible RBP-7 transcript.

Example 2 Detection Of RBP-7 Biallelic Markers: DNA Extraction

[0594] Donors were unrelated and healthy. They presented a sufficientdiversity for being representative of a French heterogeneous population.The DNA from 100 individuals was extracted and tested for the detectionof the biallelic markers.

[0595] 30 ml of peripheral venous blood were taken from each donor inthe presence of EDTA Cells (pellet) were collected after centrifugationfor 10 minutes at 2000 rpm Red cells were lysed by a lysis solution (50ml final volume: 10 mM Tris pH 7 6; 5 mM MgCl₂, 10 mM NaCl). Thesolution was centrifuged (10 minutes, 2000 rpm) as many times asnecessary to eliminate the residual red cells present in thesupernatant, after resuspension of the pellet in the lysis solution.

[0596] The pellet of white cells was lysed overnight at 42° C. with 3.7ml of lysis solution composed of:

[0597] 3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

[0598] 200 μl SDS 10%

[0599] 500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

[0600] For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5v/v) was added. After vigorous agitation, the solution was centrifugedfor 20 minutes at 10000 rpm

[0601] For the precipitation of DNA, 2 to 3 volumes of 100% ethanol wereadded to the previous supernatant, and the solution was centrifuged for30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70%ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm.The pellet was dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 mlwater The DNA concentration was evaluated by measuring the OD at 260 nm(I unit OD=50 μg/ml DNA).

[0602] To determine the presence of proteins in the DNA solution, the OD260/OD 280 ratio was determined Only DNA preparations having a OD 260/OD280 ratio between 1 8 and 2 were used in the subsequent examplesdescribed below

[0603] The pool was constituted by mixing equivalent quantities of DNAfrom each individual.

Example 3 Detection of the Biallelic Markers: Amplification of GenomicDNA by PCR

[0604] The amplification of specific genomic sequences of the DNAsamples of example 2 was carried out on the pool of DNA obtainedpreviously. In addition, 50 individual samples were similarly amplified.PCR assays were performed using the following protocol. Final volume 25μl DNA 2 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer (each) 2.9 ng/μlAmph Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10× = 0.1 MTrisHCl pH 8.3 0.5 M KCl 1×

[0605] Each pair of primers was designed using the sequence informationof the RBP-7 gene disclosed herein and the OSP software (Hillier &Green, 1991). This pair of primers was about 20 nucleotides in lengthand had the sequences disclosed in Table 1 in the columns labeled PU andRP. TABLE 1 Amplification Amplification primer PU primer RP Amplicon SEQID No. SEQ ID No. 5-124 72 87 5-127 73 88 5-128 74 89 5-129 75 90 5-13076 91 5-131 77 92 5-133 78 93 5-135 79 94 5-136 80 95 5-140 81 96 5-14382 97 5-145 83 98 5-148 84 99 99-1437 85 100 99-1442 86 101

[0606] Preferably, the primers contained a common oligonucleotide tailupstream of the specific bases targeted for amplification which wasuseful for sequencing

[0607] Primers PU contain the following additional PU 5′ sequence:TGTAAAACGACGGCCAGT (SEQ ID No. 139); primers RP contain the following RP5′ sequence: CAGGAAACAGCTATGACC (SEQ ID No 140).

[0608] The synthesis of these primers was performed following thephosphoramidite method, on a GENSET UFPS 24 1 synthesizer.

[0609] DNA amplification was performed on a Genius II thermocycler.After heating at 95° C. for 10 mm, 40 cycles were performed. Each cyclecomprised: 30 sec at 95° C., 54° C. for 1 min, and 30 sec at 72° C. Forfinal elongation, 10 min at 72° C. ended the amplification Thequantities of the amplification products obtained were determined on96-well microtiter plates, using a fluorometer and Picogreen asintercalant agent (Molecular Probes).

Example 4 Detection of the Biallelic Markers: Sequencing of AmplifiedGenomic DNA and Identification of Polymorphisms

[0610] The sequencing of the amplified DNA obtained in example 3 wascarried out on ABI 377 sequencers. The sequences of the amplificationproducts were determined using automated dideoxy terminator sequencingreactions with a dye terminator cycle sequencing protocol The productsof the sequencing reactions were run on sequencing gels and thesequences were determined using gel image analysis [ABI Prism DNASequencing Analysis software (2.1.2 version) and the above mentioned“Trace” basecaller].

[0611] The sequence data were further evaluated using the abovementioned polymorphism analysis software designed to detect the presenceof biallelic markers among the pooled amplified fragments. Thepolymorphism search was based on the presence of superimposed peaks inthe electrophoresis pattern resulting from different bases occurring atthe same position as described previously.

[0612] Sixteen fragments of amplification were analyzed. In thesesegments, 21 biallelic markers were detected The localization of thebiallelic markers is as shown in Table 2. TABLE 2 Marker Localization BMposition in Polymor- SEQ ID No. Amplicon BM Name in RBP-7 SEQ ID No. 1phism Allele 1 Allele 2 5-124 A1 5-124-273 Intron 5 72794 A*/G 30 515-127 A2 5-127-261 Intron 8 88073 A/C* 31 52 5-128 A3 5-128-60 Intron 893714 Del(GT) 32 53 5-129 A4 5-129-144 Intron 9 97152 Del(T) 33 54 5-130A5 5-130-257 Exon 11 99098 A*/G 34 55 5-130 A6 5-130-276 Exon 11 99117A/G 35 56 5-131 A7 5-131-395 Intron 12 103806 A*/T 36 57 5-133 A85-133-375 Intron 14 106940 ins(A) 37 58 5-135 A9 5-135-155 Intron 15108106 ins(A) 38 59 5-135 A10 5-135-198 Intron 15 108149 ins(GTTT) 39 605-135 A11 5-135-357 Intron 15 108308 A*/G 40 61 5-136 A12 5-136-174 Exon16 108471 C/T* 41 62 5-140 A13 5-140-120 Intron 18 134134 C/T* 42 635-140 A14 5-140-348 Intron 19 134362 ins(A) 43 64 5-140 A15 5-140-361Intron 19 134374 ins(CA) 44 65 5-143 A16 5-143-101 Exon 20 146345 A/C 4566 5-143 A17 5-143-84 Exon 20 146328 A/G* 46 67 5-145 A18 5-145-24Intron 20 150329 A*/G 47 68 5-148 A19 5-148-352 Exon 24 160031 G/T 48 6999-1437 A20 99-1437-325 Intron 8 90842 A/G 49 70 99-1442 A21 99-1442-224Intron 9 97122 G/T 50 71

Example 5 Validation of the Polymorphisms Through Microsequencing

[0613] The biallelic markers identified in example 4 were furtherconfirmed and their respective frequencies were determined throughmicrosequencing Microsequencing was carried out for each individual DNAsample described in Example 2.

[0614] Amplification from genomic DNA of individuals was performed byPCR as described above for the detection of the biallelic markers withthe same set of PCR primers (Table 1).

[0615] The preferred primers used in microsequencing were about 23nucleotides in length and hybridized just upstream of the consideredpolymorphic base. According to the invention, the primers used inmicrosequencing are detailed in Table 3. TABLE 3 Mis. 1 in Mis. 2 inMarker Name SEQ ID No. SEQ ID No. 5-124-273 102 123 5-127-261 103 1235-128-60 104 — 5-129-144 105 — 5-130-257 106 125 5-130-276 107 1265-131-395 108 127 5-133-375 109 — 5-135-155 110 — 5-135-198 111 —5-135-357 112 128 5-136-174 113 129 5-140-120 114 130 5-140-348 115 —5-140-361 116 — 5-143-101 117 131 5-143-84 118 132 5-145-24 119 1335-148-352 120 134 99-1437-325 121 135 99-1442-224 122 136

[0616] The microsequencing reaction was performed as follows

[0617] After purification of the amplification products, themicrosequencing reaction mixture was prepared by adding, in a 20 μlfinal volume 10 pmol microsequencing oligonucleotide, 1 UThermosequenase (Amersham E79000G), 1 25 μl Thermosequenase buffer (260mM Tris HCl pH 9 5, 65 mM MgCl₂), and the two appropriate fluorescentddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to thenucleotides at the polymorphic site of each biallelic marker tested,following the manufacturer's recommendations. After 4 minutes at 94° C.,20 PCR cycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C.were carried out in a Tetrad PTC-225 thermocycler (MJ Research). Theunincorporated dye terminators were then removed by ethanolprecipitation Samples were finally resuspended in formamide-EDTA loadingbuffer and heated for 2 mm at 95° C. before being loaded on apolyacrylamide sequencing gel. The data were collected by an ABI PRISM377 DNA sequencer and processed using the GENESCAN software (PerkinElmer)

[0618] Following gel analysis, data were automatically processed withsoftware that allows the determination of the alleles of biallelicmarkers present in each amplified fragment

[0619] The software evaluates such factors as whether the intensities ofthe signals resulting from the above microsequencing procedures areweak, normal, or saturated, or whether the signals are ambiguous. Inaddition, the software identifies significant peaks (according to shapeand height criteria). Among the significant peaks, peaks correspondingto the targeted site are identified based on their position When twosignificant peaks are detected for the same position, each sample iscategorized classification as homozygous or heterozygous type based onthe height ratio.

[0620] Although this invention has been described in terms of certainpreferred embodiments, other embodiments which will be apparent to thoseof ordinary skill in the art in view of the disclosure herein are alsowithin the scope of this invention. Accordingly, the scope of theinvention is intended to be defined only by reference to the appendedclaims. All documents cited herein are incorporated herein by referencein their entirety.

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1 140 1 162450 DNA Homo sapiens allele 72794 5-124-273 polymorphic baseA or G 1 ccccagagta tggactttat ttcccagaaa gccttgaggc gtaactttctgtttccatag 60 aactggtggg aaaatggcgt cgttgtttgt atccagggac caataggaacagtgtatagg 120 cgggttctaa agaactttaa ccaatccaag gtcgtctaag aggccatccgggaaagaggt 180 aggggagggg gggaaaaaaa atctagggga ggggagaaag ggggggaacctagagtcggt 240 gggggggaag cgatgtttgc ccgtcagtcg agtccggagt gaggagctcggtcgccgaag 300 cggagggaga ctcttgagct tcatcttgcc gccgccacgg ccaccgcctggacctttgcc 360 cggagggagc tgcagagggt ccatcgccgc cgtcctctgg agggcagcgcgattgggggc 420 ccggacctcc agtccggggg ggatttttcg tcgtccccct ccccccaaccagggagcccg 480 agcggccgcc aaacaaaggt accagtcgcc gccgcgggag gaggaggagccggagcctct 540 gcctcagcag ccgctggacc cgccgccctt cttccccatc tctcccccgggcctgctggt 600 tttggggggg agaaggagag aggggactct ggacgtgcca gggtcagatctcgcctccga 660 ggaaggtagg gatattttct ggggctttcg tggtctccta agggggttcttttgggagtc 720 gctgggcccg gccaaggagc agaggaagat cgcggtggtg gtccctgcggcgcccgaatt 780 cggggcctcg gcctcccggg aacccccacc ccccgcagcc gctgtgtccgagccgccccc 840 tggctgggcg gccgcacact cagcggttta gcggccgcgg tcgggggcccgggcagggtg 900 ggggccgttc cgcctccggg gccctcggcc ctcacgttgt ccccgcggcggggccagatg 960 ttgatctcgc tcccacttgt cgggtctgag cccggaacgg gacgtgggcaggggctctgt 1020 ggcgggccgg tcctgcccgc ggcccacagg ccctcctggc ccctcggtggcccccggccg 1080 gcctctcgct cggacgcggc gcgtgggggc gcggattcgc tcggccgggcgccgaggccc 1140 taggggagag cggccggccc tgcgccggac gccgggcttg ttgtgagtttcttctctgac 1200 agaaatggcg tcattgtcgt agacgggaaa ctccgtcggg tctcgacaatggggacggga 1260 agctgccgag ctgtgtaggt ggttgggttt gcggggatgg cggggccggagggagccccg 1320 agtcggcgtg tgatggttcg ggggctggcg cctggttgcg gctctttcttcggggttcgg 1380 acgtcgctcg cttctttccc tccgcctccg ccagtgcaga ggggctcctgtggtgttgac 1440 tgggtgtctt ttgtttcccc tccaggtgca gctgaacctg gtgttttagaggataccttg 1500 gtcccagagt catcatgaag gtaagattgc ctgcggaaaa cgaatctctggggttatcga 1560 attcaggaag taggaggttt cgacgaggtc gtcgtgtggg gcttccgagttttgcagagg 1620 aaggtgtgtt tggtgacttg gtgaccgagc ccttccccag agagaccggggtcagctggc 1680 gggctttgct gtggagttgg aggcagttat aggatgtctc aactgaagatgattctccca 1740 tattgaacat ctctttttgt agggaatgag aggaataacg gcatgaattaaagtgtttta 1800 aaaggtaaaa taaaaagcaa gtcaggtaat ctgaccctag aaactgaactgtattgtggt 1860 tctatcagtt acactgatga aagagaatct tatttattgt gtgaacagtactgtaaaatt 1920 ttagcagaaa actctttgac tcgtcacaaa aataatttaa ttgaaagatttaaaaagaga 1980 atttttgtga ttttttacac aattcagaga taccagttgt ttcttgtatttccagtgact 2040 tggggttata tggtttattt ttctttattg atctggttgg tctgtaaataagtcagaggc 2100 tctgttgcat tatgatgtgt ggaaaactga tggccaggtt cctcttagatgtcaatatgt 2160 taactgagtg gagaataggg tcaaaaaatt tgcttaattg ttggaagaaatgataacgat 2220 aaaaatacag gtatttttgt tacttgagag aatatgtata tgaatgctgtcttttttttt 2280 tttaaacaca tgctagaata aaaccttaga tttttgaaaa gtctggattcaactggtgga 2340 acttagacac tagcttgatt ttttcatcca ttacaaattc agaagtgcttgaaaagggta 2400 gagtgttcac aagtgtagaa atgcaacaga tagcatgtta gaatatcaacgttgtctact 2460 tttgacagtt cctttgaact atttaaaaaa atatttcact tgtcctcagaatcaagactg 2520 ctttgacaat tttgcatcca aattaaaatt tgtaatactt gtggttgtagaaggtaaaag 2580 ctgtggctac ttaaatcttg gtctactata cagagtaatt aagtttagtctaattttttt 2640 aaggttttct ctttgagttt gggatgctgt aaactacctg aaacagtttattcctatctc 2700 atttcatgaa gcatagacat gtttcaggaa tgaatccctt ttgttagtcacaaatcctag 2760 ctataactct catcacctgt gtaaaacagt ggtgaaagtg catttattaggtaactcgta 2820 tatgtacctt gatgttgggt gagaatttct taagaaaagt tttggtttgagtaagaaata 2880 ataagttcag aagccgaata aataaaaggt ttagtttgga gtaaatgtaatgtgtcacat 2940 ttcccagtgt tgtagtgctt gtcacttctt aggtgctcta taaatgtttggttaaaagaa 3000 atgtagttaa gttgaaagtc tgagattaaa atgttttttt tcttcctttcgtttcctttt 3060 tttttttaag tagtctgttt aaaaaaatga aagtatgttg gaaactttagccttaaaagt 3120 ttcttggtaa tactagccaa atgcaaattt tttggtatct ttttagaaatactaatttta 3180 agtgctcagc attacattct actgcatttt ttaaaaaggt gcttttaacgttaaaaggaa 3240 aaagttacta aaataaattt ctaacttcat acaagtactt cctgttattataagtatagc 3300 aaatctcaga gaatttgcta gtaattatta gtactaaaaa cttagcttatttcttacaac 3360 tttatttctt atgtgataaa agatgagaag cttcttgtaa tttgtattttgctttagcaa 3420 aaattgcaat tcagaatcta tttaatgttg agtgtattag gcctaaattttgtttctttt 3480 tctgtgtgtt cccaatttct ttcgttcttt tttatttttc tttttgatatagagtctcac 3540 tatttcaccc tggctggcac gatctgtaac tgctgcctcc tgggctcaaataatcctccc 3600 acctcagcct cctgagtagc tgggactaca ggcacacctg gctaatttttgtagtttttg 3660 tagagatgag gttttgccgt gttgcccagg ctggtgtcga acttctgggctcaggtgact 3720 ctcccgcctt agcctcccaa agtgctggga ttacaggcga gagtcaccgtgcctggtgta 3780 atttctgtat tacgatgatc attgagctac agaaggtttt atgccatttattggatctta 3840 ccaaacatat ttgtacttgg gagcaagctt ttaagtttga attgtctgttgtattaacga 3900 caacaaatct ttaaatttta ttaagtgaca gtttattgag gtgattgaaagttagattat 3960 gtagtaatac tttttaggtg ttgttttaaa gttgaagtgt tgtatgttttagcagttttc 4020 ccatgtacta cctttaagaa tttatgtaaa caaatacatt cccattactgtctaatatat 4080 gctatgttac ttttttcaca gtatgttttt gaggaatatg attcgtaatacataaggaat 4140 ttgtgctgta acatagatcg tgatgttggt gttatgttga atttatttttcatgctcttg 4200 atagactttt aaaaatcagt ggtgccggct gggcatggtg gctcacgcctgtaatcacag 4260 cactttggga ggccgaggta ggaggatcac ctgaggtcaa gggagtttgagaccagcctg 4320 gccagcgtag taaaaccctg tctcttctaa aaatacaaaa ttagctgggcatggtggcac 4380 acacctgtaa tcccagctat tcggaaggct gaggcaggag aactgcttgaacccaggagg 4440 cggaggttgc agtgagctga ggttgcgcca ctgaactcca gcctggcgaaaaaagtgaaa 4500 ctctctctct caaaaaaaaa aaactaaata aaataaaata aaaatcagcagtaccacatt 4560 ataaggaggt ggtagattat taaagtttgt aaataattcc aagacttatttttattatgt 4620 caggttgatt tttacttttt ttttttaaga cggacttttg ctcttgttgcccaggccgga 4680 gtgcagtggt gcgatctcgg ctcactgcaa cctctgcctc ccaggttcaagcagttctcc 4740 tgcctcagcc tcctgagtaa ctggaattat aggcatgcac catcatgccgggctaatttt 4800 gtgcttttag tagagacaag gtttctccat attggtcagg ctggtctcgaactcctgacc 4860 tcaggtgatc tgcccgcctc ggcctcccaa agtgctggga ttacaggcgtgagccactgc 4920 gcccggccga tttttacttt ttttaaactc aaactttttt ttcctgtatctatattgtac 4980 atgttattaa cgtcttttat tacgtagcag taaattttca attaaaatgggctggttggg 5040 ctggatatac cagaatcttt agaggaggaa tgatttagat gattatactttggcgggcat 5100 ggtggctcat gcttgtaatc ccagtacttt gggaggctga ggtgggtggattacttgagg 5160 ttgggagttc aagaccagcc tggccaaagt gatgaaaacc ccgtctctactaaaagtata 5220 aaaaaattag ctgggcatgg tggcgcatgc ctgtattccc agctactcaggagactgagg 5280 taggatcgct tgaacctagg aggtggaggt tgcagtgagt cgagatggcaccactgcact 5340 ccagcttggg tgacagggag accccatttc aggaaaaaaa aaaaaaaaaggtggttatat 5400 ttagaatcca cgtagattga attttccaga tgtacttttc tttcttcaacaatgattggc 5460 ttaggccaca tgaaaaaata atggattgat tacaaattcc cttttttaatgccctgtgtg 5520 tgtgtacatg ttctctctct ctctccccct acctctacct ttgtttgtctccctatccac 5580 tctgtcagtt cgttcccccc tctctgttct tttccttccc cctcttccttctgtctcacc 5640 cctttcataa gtcattcatt taacaacaac ccgtttcata tatcaggtaatatagaatga 5700 aggtgggtga ggcatctgac ctgctttcag ggagcccaca gtgtagcagaagatgcagat 5760 aaataaagca aaactcctag catatctatt tatgagtatt ccaacataaaataattgtag 5820 gaaatgctat gagaggacag aagaggacag ctaaatcaga gagctatggaaagttttctg 5880 gtggagttga catctaagct gagtgcgaag gcttttgcta ggcaaagaagggaagaaaag 5940 tttttcagat agaggataac gtactggaaa atgaacgaaa caaggtgaagtatctggaaa 6000 ctgtaagtaa tttggtgtga ctgaattaac ttaaagcatg atatgtttgggtaggaataa 6060 aagggcagtg gtgggagatg aagctgagtg agggagtcga aaattttcttggccaggcat 6120 ggtgtctcac acctgtaatc ccagcacttt gggaggttga ggcaggtggatcacctgagt 6180 tctataccag cctggccaac atggtgaaac cctgtttcta ctaaaaatacaaaattagca 6240 gggcatggtg gtgtgagcct gtaatgccag ctattaggga ggctgaggcagaagaatagc 6300 ttgaacacgg gaggtggagg ttacagtgag ccgagatcat gccattgcactccagcctgg 6360 gtgacaagag caatactctg tctcaaaaaa aaaaaaattt tttcttgaaaactacatgga 6420 accattgaaa gttgttgaga attgcttgaa cccaggagct ggaggttgcggtgagctgag 6480 atcaggccac tgcaaaaaca aggggagagc tgaatgtaag gatggaaattccaggtaaga 6540 agtccaagtg gaaaatgaat aagggcctga actaatgcat tggaagtggagcagagagga 6600 tagatctgag cctttttgaa gtagaattca gggccgggca cagtggctcacacctgtaat 6660 cccagcactt tgggaggctg aggtgggtgg atcatgaggt caggagattgagaccatcct 6720 ggctaacaca gtgaaacccc gtctctacta aaaaaataca aaaaatagaaattagccagg 6780 cttggtagcg ggcgcctgta ggacgctgag gcaggagaat ggtgcgaacctgggaggcgg 6840 agcttgcagt gagccgagat cacgccactg cactccagcc tgggcgacagagcaagagtc 6900 tgtcttaaaa gaagtagaat tcaggaccta gtaactcatc ctggggtaggaagaagatgg 6960 aggttttttt cttaggaaat tagatagatg ttactgtcat ttgaagcatgctgtcattgg 7020 acctgctagt taagatgtaa gagtaagtag gtaggatgaa gtcagaggcttggttaatct 7080 ggtgtgaatg ttaacctcat atctatgata tttgaaggga tttaagatccataacatggc 7140 aaattttgtg ctataaattt gtggtccttt agatgaatag gtaatcttgtaaaatactaa 7200 catgttttct gtccaaattg ctaaatcttt gtttattgta attaaaaaaaatactaggtg 7260 agtacctatg tcactgaaga atgtttttgc caagtcagcc agtgaattcagtcattgaac 7320 acattgtgta ctttccttgt accggacatt gttctgggtt ttgaggatgcagcagtgaat 7380 agaacaagag ttcttaccct cgtggagttt actttctact atggtggacacacgttaaga 7440 aaaaaaataa tgctaagtgc tttgaagaga aatgatacgg aatcagaataagaggttaga 7500 gtgacagagg gtgggttact ttagattagg tgataatgga gggccttttcgagatgacat 7560 ttaagtgtgc tgtgaaacaa gtgatagagt gagccattca ggtttctgggggaggagcat 7620 tccctgcaga aaacaagtgc aaataccctg agatggagtg tgcttggcatgtttgagtgt 7680 gatacattta aggaacatta aggccatcag ggctggagtg gtaggagaggaggtcagaga 7740 gggtcacaaa gggccagtac aattgtaggg ccttatagga catggtaaggacttgagtaa 7800 aatctgaatg tgttaggaac cctttgaagg gtgttgagtg aagtgatgtgataaacattt 7860 tcaaaaatgt ttgactgctg ggtagtgaac acatcatggg ttcgaacagatcatgggaac 7920 acaagagtgg aagtagggag atcagtcaga aggctgtcaa gctagtcctggggagagatg 7980 gtggtgagga gaaatagtag tattgggaat atatttgaag atggggatgattggatttca 8040 atggattggg tgtgaaggga aaattctact tactgcaact tgcacatgtatgtcctccca 8100 caagtgccct ccctcctttt cttttgagac ggagcctctc tctgttgcctaggctggagt 8160 gcagtgatgt gatctcggct cactgcagcc tctgtctccc gagtttaagtaattctcttg 8220 ccccagactc ccctgtagct gggattacag gcacctgcca ccacgcctggctaatttttt 8280 ttgtattttt tgtttttttt tttttagacg gagtttcgct cgttacccaggttggagtgc 8340 aatggcacga tctcggctca ctgcaacctc tgcctcccag gttcaagcgattctcctgcc 8400 tcagcctccc aagtagctgg gactacaggc atgtgccacc atgtctggctaatttttgta 8460 tttttagtag agacggggtt tcaccatgtt ggccaggctg gtctcaaactcctgaccttg 8520 ttatccacct gcctcggcct cccagagtgc tgggattaca ggtgtgagtcacctcgcccg 8580 gccttttttt ttgtgttttt agtagcaatg gggtttcacc atgttggccaggatggttct 8640 gaactcctga ccttaagtga tccgcccgcc ttggcctccc aaagtgtgagccactgcacc 8700 tggccgtcct cccttttttt aaaaaagtct ttttgctgtg attagttggtagatgggatt 8760 cttggaactg agtatgacca tgaaggcatt gtcaaatctt agccttctcagtgagcagtg 8820 aacattgagt tgagcaatcc tttcacttgt ttttttaatt aaaaaaaatgagatataatt 8880 cacataccat aaaattcacc cttttatttt attttttttg agacatggccacagtctgtt 8940 gcccaggcag gagtgtagtg gcgcaatcac agctcactgc agccttgatcttcagtgggc 9000 tcatgtgatc ctgcttcaac ctcccaggta gctgggacca gaggtgcacggcaccatgcc 9060 cagctaactt ttaaattttc tgtggaggtc tcactgtgtt gcccaggctggtcttgaact 9120 cctgggctca aatgatcctc ccatcttggc ctaccaaagt gttgggattacaggcatgag 9180 ccaccgcgcc tcaccaaatt caccttttta aagtgttcat aaatttctagtattttcaca 9240 agtttgtgca tccatcacta ctatcttttg aaaacttttt tgttaatttcttttgtttgt 9300 ttttttagag atgaggtctt gctgtgttac ctaggctggc cttgaactcttgtggtcaag 9360 tgatctttcc ccctcaactt cctgagtagc tgagacttca ggcacgttacttatccctag 9420 ctatctgtca ctgctattta actccagaac atttccaaat tcccaacaagaaacttctta 9480 tccattagca gtcattcact gttctccctt tcttttatcc ctccagtaactaatctaaat 9540 tttttttttg agaagagttt tgattctgtc acccaggctg gagtgcagtggctcgaccat 9600 ggctcactgc agcatcgacc tcctgggttc aagcgatctt cctgcctccagctcccaagt 9660 agatgggatt acaggtgtgc gccaccatgc ccagcaaatt ttggaattttttgtagtagt 9720 gaagtctcac tatgttccct agcctggtct ggaactcctg agctcaagtgatcctcttgc 9780 attggccccc aaagtgctgg gattacaaga ttcagacact gagcttagcatttactttct 9840 gtctataagg atttgcgtat tctgggcatt ttctgttaac ggaatcctgtaatacatatg 9900 tggccttttt gtgtctgatt tcttttactt cccataagtg aaagaagtagcaagtgttga 9960 ccaggcgcga tggctcacgc ctgtaatccc agcactttgg gaagccgatgtgggtggatc 10020 acgatgtcag gagttcaaga ccagcctggc caagatggtg aaaccccatctgtactaaat 10080 aaaaaaatta gccgggtgtg gtggtgggcg cctgtaatcc cagctactcaggaggctgag 10140 gcagagaatt gcttgaacct gggaggcaaa ggttgcagtg agctgcgatggcgccactgc 10200 actccagcct aggtgtcaga tcaagactcc gtctcaaaaa aaaaaaaaaaaaaaaaaaaa 10260 aggagtagca agtgttaata ctatgctctt ttttattgct gaataatagtccatcttatg 10320 aagatacgac attttgtcag tctacttatt tggaggacat gtgggttatttctgcttttt 10380 tgcttattgt gagtaatgct gctatgagca tttgtatgta agtttctgtgcgaacatgta 10440 tttttaattc tcttgggtcc tgggagtgga cctttcagtt cttagggtactaatattacc 10500 tgtgtgataa cctggggagt gctgagtatt taacttcatg tattttgtgttgtggcaagt 10560 ggccaccaag gagttggaca tttaaaaaat taattcaata tttattgaatacttattatg 10620 tattggtctc tgttctagtc agttggaata tgtcagggaa caaaacaggtagaaattcct 10680 ggctctttgg atctcttagg agtacaggaa actctggtat tcaaggtttccgtagaaaat 10740 aatgtgcttt tagaaatcat aattttcgtt ctaaaattgg agacagcattcagatttggg 10800 gaagaaaaaa atgagtttat ggaagtatat aaacagttta ctagaaaataaaaagcaggt 10860 ctactaaggt cagcatagac aaactatctt catgttcacc ttaccatagtggctgttatt 10920 ttaagctggt taatagaagc aagggacata atgaaagagt caaaaagggaaaagttttaa 10980 aacaatactt ggcttactct gagcatagtt tcccctttcc ttaattaacgcttgctcata 11040 tattactcag aaagtccaag tgtaggcttc agaggagagg agccatagatagctttcatc 11100 agattcttga tgaaacccac agtacaaatg ttaagaaaca cagctctgtggtatcatctg 11160 ttgactgatc tgctgctaat ctatttaata ggaagagttg tttctatatccttctacttt 11220 taccattaaa gaaaaagtaa tcaactagtc actgttcatt ttattttcaaatttattttt 11280 gcttaaatca ttgcagaatc agaaaaaaat ttttattata ttgtttctgaaatgttaaca 11340 tttaggtgaa atgcttaatc aggttgagta tcacttacct gaaatgcttgggaccagaaa 11400 tatttgggat tttttcagat tttggaatat ttgcatttat atgcttagtatttgaacatc 11460 ccaaatctga aaatccaaaa tgttccagtg agcatttccc tttggtgtaacatgaacact 11520 gaaaaagttt cagtttttgt agcatttctg attttttgtg ttttacgtatgtatatgtat 11580 atctgtatct tgtttttttg tttggttgtt tgagacagaa tcttgctctgtcacccaggc 11640 tggagtgcag tggtgcgatc tcagctcact gcaacactcg gctcccggattcaaacgatt 11700 cttctccctc agcctcccaa gtagctggga ttacaggcgc ctgccaccacacctggctaa 11760 tttttgtatt tttagtacag acgaggtttc gccatgttgg ccaggctggtctctcgaact 11820 ccagacctca ggtgatcctc ctgcctcagc ctcccaaagt gctggaattataggcatcag 11880 ccaccgtgcc cagccttata taaatatatt attatttttt tgagacgaagtcttgctgtg 11940 tcgcccaggc tggagtgcag tagtgcgatc ttggcttact gcagcctctggctcctgggt 12000 tcaagtgatt ctcctgcctc agcttccaga gtagttggga ttacaggcgtgtgccacaac 12060 acctggctaa ttttttgtat ttttagtgga gacgaggctt caccatgttggccaggctgg 12120 tctcgaactc ctgacctcaa gtgatccgcc cgcctcagcc ttccaaagtgctggaattac 12180 aggtgtgagc cactgcaccc cacttatttt tgagttaggg atactcaatgtgaattgctt 12240 gaaatgttta cctcgttgaa ttcctaagaa gaatttgaat tttttaaatttataactagc 12300 ctttgatcca tggaaacatt ttataaaata atttccaaaa taatttcctggaaatctgga 12360 attgtagtct gtagcaaatt gggattattt attaatttaa tttaatttaatttatgagat 12420 cagagtcttg gtatgttgcg ttggctggtc tcgaactcct aggcttgagtgatccttctg 12480 cctcagcctc tctagtggct ggaactgtaa gtgcacacca ccatggcacattggatatta 12540 tttatgaaac tatttattac aaatgttagt atatgcttac tcttaccttttgcatattca 12600 attatttact ctaatcgggt tatctaaggc aagaatagta tctaactgtgaataaccaga 12660 tatgcttagc tttaggatac agttagacgt aagtatagaa ttcaacatccctgtaactaa 12720 tgtctttcca gattaatgtt agtgttgata gtaaggtggt agaacgggctaattctctgg 12780 gccattatta ctgatttata aggtagaaaa tagggtgtat cactttaaagttacaaattt 12840 acatttataa ggaagaaaat agggtatatc acttttaagt tacaaatttacctgtcatca 12900 attaagagaa taataattag gcagtaggtt tataccatta aaatgtgtgagattacttac 12960 actatatctt ggacagtggg acagataatt tatttttttg gagacatagccttgttctgc 13020 ggcccaggtt ggagtgcggt ggcgcaatca tagctcactg cagcctccagctcctgggct 13080 caagtgatct tcctacctca gtcacccaag tagctgagac tacaaataatgcacaccacc 13140 atgcctcgct aaattttttt gtatttttgg tagagacggg gtctccttatcttgcccagg 13200 ctagtcttga actcttgagc tcaggtgatc ctcccacctt ggcctcccaaaatgttggga 13260 ttacagatgt gagccaccaa gcctggcctg gacaatggga cagatactttatatgtagac 13320 ttttctcatt taagccattt ttctctagtt tatagataaa tttttggcaatgtgacaact 13380 agaatatctg aaatcctata taaaagttct attaatgcta tctgctaattgataaccttt 13440 tgatttcagg gtataattgc ttatgagaac atagtcattg ataactttagtaagtttcat 13500 tgaacatcat attttaacaa tgcacttcaa tagcaaatga agattataaactaaaacctt 13560 tgagcaagtg gtatttaaag aaaggattta ctttatattg atagccaaaataatgtatat 13620 accgaaatat atataaatac gtatttataa acatatttta ctgatagtaaaatgttatat 13680 atattgttat atatcattta agataattta tatgtaaatt aaatataaaccagatttctt 13740 tattccagga accttgctgt tgtttctaac ctttcttttc ctgctacttagtgatgcaga 13800 agcttttctt ggtaaatcta cttgtcccct ctcttaattt actgcttgaatatgactgtg 13860 aaaactgtct ttgtttaaag tgcccagtaa acattgtagc ttcatgattaagagcatatg 13920 gcttggaaag tcagtggtct tatttccagt cctttttatg tcctttacttggctgctgag 13980 gtttgggtaa agttactgtt tttgaaatga aagtaaacat cagtttcaatttttgtaaaa 14040 tgagaataat aacagctacc ttgtaaagtt gtgtaaatat gaaatgaaataccatattta 14100 aaatgcttta cactgtgctt ggcatagttc tgagcagtta gtacgtggcagttgtattat 14160 tagaggaagc ctgtcttgtt tttttttaaa taagctgata gagtgaggattcttttaatc 14220 aagactgttt gggattgaat tgccactcct gcttaccaga gtgtaggcagtttttcttaa 14280 actttccaag aagactggtg tcctcatcta aaatacgaaa tgcttacagtaattgcctca 14340 tggggttgtt tggggtgact aaatgtagta ggatttacta catagtaagttctcaataca 14400 ttgtagctat tattattagt tcggtagaaa gaatgtgcag attcttatgagtttaagtag 14460 gctttcgggg agatagattg actctggtct tttaaaagtt aattttgaagttgcagtttt 14520 gtgattaagc cttaaatctg ttattctttc cttctgaaat ccttaaaaacagaatgttta 14580 gtagaaggtg ataaccagat ttctttattc caagaactct ttgctctcatgtctaacctt 14640 tattttcctg gtacttactg atgcagaagc ttctcttagt aaatataatacatctcctct 14700 ctcctaattt gctcccgtct ttccttgtaa gggaaaagta aattttacttccaagcctag 14760 agggttattt atggattagg tgaactactg aagatactta ttttctggataagcatccat 14820 ctgtatagcc ttttatgtat ggcaaaaatt gttttcattt cttgatcagaatactgttct 14880 gatgtggtgt agtcagccac ctgaagctga tctagcatgg gcagcctaggcaggtagggc 14940 gaatgactgt aggagccctg ctaaacccga gtctctactc cagagaggagttaaaaaaag 15000 ctgaacaagc ctgaacacgg aggagccact attgctgtca aagttaagtgaagcagcttg 15060 gcttatgtct atttcagaat aaaaaaaaaa aattcaactt aggcatgcacggtgggtcat 15120 gcccgtaatc ctagcacttt gggcgtttga ggctggcaga ttacttgaggtcaggagttt 15180 gagaccagcc tggccaacat ggtgaaacct tgtctctact gaaaatataaaaattagcca 15240 ggcatggtgg tatgcctgtt tgggaggctg atgcaggaga atctcttgaacccgggaagt 15300 ggaaattgca gtaagctgag atcgcaccac tgcactccag cctaggtgatagaatgagac 15360 tgcatctcaa aaattcaact tttcacattt tcagttttat cttgataaggatacctcatc 15420 aaagaaccct tttcttttct tttcttttct ttttcttgaa gcagaatcttgctgtgcatc 15480 ccaggctgga gtgcagtggt gcaatcttgg ctcagtgcaa cctccgcctcctaggttcaa 15540 acgattcttg tgcctcagcc tcctgagtag ctaggactac aggcatgcgccaccatgctc 15600 ggctaatttt ttgtatttta gtagagatgg ggttttgtaa tgttacctagggtggtctca 15660 aactcccgag ctcaggtgat cgcctgcctt ggcttctcaa agtgctaggattacaggtgg 15720 gagccaccac gcccagcccc atttctgttt tttttaatct gaatattctttgctaaagtg 15780 ttgttgtttt ttttaaaggc aagccagaga gcttttggca tttagtaagtcacctattcg 15840 tgctttgctc taacttggag aaatattttt ctaaactagt tcttctagcaaaattaaaga 15900 aatttttatt atgaaagcaa taaagatttt ctgtaaacat taaaaattacatagtagtat 15960 tcagtggaca gtagaagtct tacttcctgg taacttgatt caacagtttttgggtgtatt 16020 cttccacacc tttttctttg cacatatgaa tcaaacatga gtatttattttgttatgttt 16080 gactgtattg aaatgggctt atgctatgca tattgttcgt aattttcttttcttttcttt 16140 tctttgagac ggactgtcgc tctgtcacca gactagagtg tggtggcacgatcttggcta 16200 actgcaacct ctgcctcccg ggttcaagca gttctcctgc ctcagcctcctgagtagctg 16260 gaactacagg tgcgcgccac cacgcccagc taatttttgt atttttagtagacagggttt 16320 caccatgttg gccaggctgg tcttgatctc ttgagctcgt gatttgcctgcctcggcctc 16380 ccaaagtgct gggattacag acgtgagcca ccatgcctgg cccataattttcttttacat 16440 tcttctatat cagtacatat tacacaagtc tgtgtcattc ttcttgatggctttagtaag 16500 attttatttg cattcaacat gttatttttt ctacaaccaa ataatttatatagggcaccg 16560 gtatgtttct ttaagttttg caggtatgta gcagtgctta gtattcagtaggtactgttt 16620 ttgtttgttt gtttgtttgt tttgtttttt gtttttgaga cagggtcttgctctgttgcc 16680 caggctggag tgcagtggca cagtcttggc tcactgcaac ctctgcctcccgggttcaag 16740 agattctcat gtttctgcct cccaagtagc tgggactaca gtcccgtgccatcacaccca 16800 gctaattttt gtatttttat tagagacagg gttttgccac gttggccaggctggtctcaa 16860 actcctgacc tcaagtgatc tgcctgcctc gacctcccaa agtacaggtgtgagccactg 16920 tgcctggcct tcagtaagta ttgttaactt atttggatgc ctagtactagtaggtggcaa 16980 atagtcccag tacctattat atatacaaag cttcttatag tcctatgaattattattatt 17040 attattatta ttattattat tattattatt attgttttga gacagggtcttgctctgtca 17100 cccaggctgg agtgcacagt acaatcacgg ctcactgtag ccttgaccttctgggctcaa 17160 gcgatcctcc cacctcagcc tctcgagtag ttgggactac aggtgtgggtcaccataggt 17220 tgatttttta tattttctag agatggggtc tcactatgtt gcctaggctggtcttgaact 17280 cttgccctgt gaattattgc agccaccaac tgttaaatat cattgcatgacattgttact 17340 aaaaggtaat ctatgaggat tagtgaggga gcatccctgt gctatatggctggttctaaa 17400 aaagcttatg ctgttctttg ggatccctgt tagcattgat tagacaggttaattttgggg 17460 gccgggtgcg gtggcttata cctgtaatcc cagcagtttg gtaggctgatatgggtggat 17520 tacttgaacc caggagtttg agaccagcat ggacaatgtg gcaaaaccctgcttctacaa 17580 aaaagtttta aaatagccag gagtggtggc ctgtgactgt atttccagctactcaggaag 17640 ttgaggctgg gaggattgct tgaacccagg atgtcaagtc tgcagtggagctgtgattgc 17700 agccactgta ctccagccta ggtgacagag caagaccccg ttttaaaaacaaaatcaaaa 17760 acacgttaat tttggaatgg atctctatag gaagtgtccc cagcatatgctcaaagtcag 17820 aatatatagt ttataaggaa ttctttaacc gtacagttat atggcacattacgtttttaa 17880 agttccataa tcattagtta tatctaataa catccctttg aggcaggtcagcaacatttt 17940 tcccttttta tggtcgaaga agcaggtgta aagatgttga gtgatttgcccacaggcaca 18000 cattaaactg atcacagagc ctgttctttt gttagtaaac tgaaccatgctgcctctcta 18060 ctagttatta taaataaata ataaagttgt tgccatttag tgactttttgatggctttat 18120 tgaatagtaa gtgtagttta caaacctttt gcctacttac tttgttgaaaaagttttaaa 18180 tttagaattt acttgtatca tggtatgaaa catttttatg taaatttccctgtttctttt 18240 tttttatttt tttttattga tcattcttgg gtgtttctca cagagggggatttggcaggg 18300 tcataggaca atagtggagg gaaggtcagc agataaacaa gtgaacaaaggtctctggtt 18360 ttcctaggca gaggaccctg tggccttccg cagtgtttgt gtccctgggtacttgagatt 18420 agggagtggt gatgactctt aaggagcatg ctgccttcaa gcatctgtttaacaaagcac 18480 atcttgcacc tcccttaatc catttaaccc tgagtggaca cagcacatgtttcagagagc 18540 acagggttgg gggtaagtaa ggtcacagat caacaggatc ccaaggccgaagaatttttc 18600 ttagtacaga acaaaaggaa aagtctccca tgtctacttc tttctacacagacacggcaa 18660 ccatctgatt tctcaatctt ttccccacct ttcccccctt tctattccacaaaaccgcca 18720 ttgtcatcat ggcccgttct caatgagctg ttgggtacac ctcccagacggggtggcggc 18780 cgggcagagg ggctcctcac ttcccagtag gggtggccgg gcagaggcgcccctcacctc 18840 ccggacgggg cggctgaccg ggcggggggc tgaccccccc acctccctcccggacggggc 18900 ggctggccgg gcagaggggc tcctcacttt ccagtagggg cggccgggcagaggcgcccc 18960 ttacctcccg gatggggcgg ctggccgggc ggggggctga cccccccacctccctcccgg 19020 acgggtggct gccgggcaga gacgctcctc acttcccaga cggggtggttgccgggcgga 19080 ggggctcctc acttctcaga tggggcggct gccgggcgga ggggctcctcacttctcaga 19140 tggggcggct gccgggcgga ggggctcctc acttctcaga tggggcggttgccaggtgga 19200 gggtctcccc acttctcaga cggggcggcc gggcagagac gctcctcacctcccagacgg 19260 ggtcacggcc gggcagaggc gctcctcaca tcccagacgg ggcggcggggcagaggtgct 19320 ccccacatct cagacaatgg gcggccgggc agagacgctc ctcacttcctagatgggatg 19380 gcggccggga agaggcgctc ctcacttcct agatgggatg gcggccgggcagagacgctc 19440 gtcactttcc agactgggca gccaggcaga ggggctcctc acgtcccagacgatgggcgg 19500 ccaggcagag acgctcctca cttcccagat ggggtggcag ccgggcagaggctgcactct 19560 cggcactttg ggaggccaag gcaggtggct gggaggtgga ggttgtagcgagccgagatc 19620 acgccactgc actccagcct gggcaccatt gagcactgag tgaaccagactccgtctgca 19680 atcccggcac ctcgggaggc tgaggctggc ggatcactcg cggttaggagctggagacca 19740 gcccggccaa cacagcgaag ccccgtctcc accaaaaaaa tacaaaaaccagtcaggcgt 19800 ggcagcgcgc acctgcaatc gcgggcactc ggcaggctga ggcaggagaatcaggcaggg 19860 aggttgcagt gagctgagat ggcagcagta cagtccagct tcggctcggcatcagaggga 19920 gaccgtggaa agagagggag agggagaccg tggggagagg gatagggagagggaggggga 19980 gggggagggg agcccctgtt tcttaaaaat tagaaaaaat caggctgggagcggtggctc 20040 atgcctgtaa tcccagcact ttgggaggcc aaggtgggcg aatcacttgaagtcaggagg 20100 agttcatgac cagcctggcc aacacagcga aaccctgtct ctactaaaaatacaatacaa 20160 aaattagctg tgcatggtag catgtgcttc tggtcccagc tactcaggcgctgagacagg 20220 agaagtgcct gaacctggga ggcggaggtt gcagtgagct gagatcaggtcactgcactc 20280 tagcctgggc gacagagcaa gatgcaagac tctgtccccc tccaaaaaaaaaaaaaaaaa 20340 aaaaattaga agaaatcata tgaaaaaaca taaaaatgaa cgacagtggacttagttctt 20400 ctgggagaac tttgctagct tgaaaattag tgtcaagctg gacaggtggcttgtgcctgt 20460 aattccagct acttgggagg ctgagacagg aggagcttga gcccaggagctcaaggctgc 20520 agtgagcctt gattgcacca ctagattcca gcctgggcga cagagcgagactctatctct 20580 aaaagaaagg aaaattagta ttataaatta gaaaattaga ggctttgtacaaagctttga 20640 agagttaata ttttaaagta ggagaaaaat gaatcctctg gttatattgccacaagtaat 20700 tttgcgttga aacactgtgg cccatatagc ctgaagagac tttgaaaggtcatttgagcc 20760 cttgagttca gaacctatct cttctgccaa gtgatgacac agaatatgatctagctcaga 20820 aaagttttac aatctgggtg gtttgtctct ctttttctct ctctctttttctttggctga 20880 agaatcttta gaacagtcaa ccttctctta aattgcaagt ttcctttggccatttgaaac 20940 attttctttt tttcttttct ttttcctttt tctttttttg taaagagacagggcctagct 21000 ctgtcactta gactggaatt tagcggtgtg atcataattc actgtagccgtgaactcctg 21060 gattcaaggg atcttcccat ctcagtctct tgagtagctg ggaatacaggcttgcgccac 21120 cacattcagc taagtttttt atttttttgt agtgctgtgg cctcactgtgtttcccaggt 21180 tggtcttgaa ctcctggcct caagtgacct tcccttgttg gcctcccaaagcactgggat 21240 tacagacatc agccactgca cccagccaga cattttctat ttacttgtatggacttaatt 21300 ctgtctatcc cattccccct tcccccatta taattttttt tctctgtaatttgtaggtac 21360 aagttctttc tctcactgaa aagctttcca attgtagttt ttctgcattgatggaggtag 21420 tgaaggggag gttcaggtaa tttacaacta ttgtaaacag tttttatttggcatggtaca 21480 gtgtctaatg cctgtaatcc cagcactttg ggaggctgag gcggggaaaattgcaagttt 21540 actttgagtt tgagactagc ccggccaaca tcgcgaaacc ctgtctctaccaaaaataca 21600 aaaaaattag ccgggtgtgg tggtgcacac ctgaaatccc agctgttctggaagctgagg 21660 cgtgagaatc gcttgaatcc gggaggtgaa agttgtagtg agctgaggtcacaccactgc 21720 actccagcct gggcgacaga gggagagact ctgtcttaaa aaaagagattttatttaagg 21780 gatcatacag agccctaaaa ttatatattc acatttggaa tgttaagcagatgctttgac 21840 atgatatatt ataaatctgt aataataaat agtagttaat cctcatatactttagtaatt 21900 agcacagtcc aatttttttt ttttttgaga tggagtctca ctctgttgcccaggctggag 21960 tgcagtggta tgatctcggc tcactgcagc ctctgccttc catgttcaagtgattctcct 22020 ggctcagcct cccgagtaac tgggactaca ggtgtgtgcc accacgcctggctaattttt 22080 tgtattttta gtagagacgg ggtttcactg tcttagccag gatggtctcgatcttctgac 22140 cttgtgatct gcctgtctca gcctcccaaa gtgagccacc gtgcccagccagcacaatct 22200 tattttttag taggcatgta atttctaaat ttgtctttat tgctaaagtaacatcccatt 22260 tatctcaaac taactgtcat cagtcttgtc ttgttctgaa atagcattaaaatatttatc 22320 acaccagcct ctttcctggt catcatgttc tttatgctgt aattattattgtttttgtta 22380 ttatcatatg ataattttgc cactgcttat tcagtgctta atatatgtcagtcttttctt 22440 acattatatg tagttttttc ttatttgaag acagagtttc ttgctctgttgcctgggcag 22500 gaatatagta gcgtgatcat ggctgactgt aaccttgacc tcttgggcccaggtgatcct 22560 cccaccttag cctcctgagt agctgggagt acaggtgtcc accaccatgcctggcgaatt 22620 tttaactttt ttcatagaga cagggtctca ctatgttgcc taggctgatcttgaactccc 22680 cggttcaagt gatccccctg cctctgcctc ctaaagtgct gggattataggcgtgagtcc 22740 ccgtgcctgg cccatttaaa attttaattc tcaaaatacc ctgagaggtacatagatata 22800 ttcttatttt acagatgaaa aactcaaggc cgagagagat tatataatttacctaactaa 22860 ggtcatgcca ctagtaagtg gcatagtgag gatttaaacc taaactacttcaaaccagag 22920 ctcctactaa tattaatgct gtttctgcct ctttaatata tatctttacttggatatcta 22980 gttttcttag tacacagaac atttagaagt gccaaatact gtgtgagttgttagttgtaa 23040 gtgtatgtgt gtttttatgt gtgtatcgag agggaggagt tttgaacaaatccggaagtc 23100 agaggtctga cgtgggtctc actgggcatt tcttcttcag gctctaggggagaattggtt 23160 tccttgcctc ttttccagct tctagaggct gtatttcctc ctttgcctctggtctctcct 23220 attttcaaag ccagcagcct ggtgtcttca aatctgtctc tgacactatctctcctgcct 23280 ccctctttca cttataagta tccttgtgtt tatattaaat agggctcacccagataatac 23340 aagctaatct ccccctttca aggtcaacta attagcaact ctgattccatgtcagcttta 23400 aatttcccct tgatagataa cattttcaca tgttttggag attcggatgtggtcattttg 23460 tggggagggg gagtgcattc tgcctaccag agtttcctta gtgtggcaaaatgatttagc 23520 ttcttaggcc tcgctataaa atgagattag tatttactat ttaccacaaaggaattttat 23580 gaagatggaa attacgtact aatacagaca ggccatgcat agaatacacttaattagcag 23640 atgctttctg cttgatctta tctgctaggt atatgcttgt ggtgatgggtagtctccaag 23700 gtaaccactg ggtttgataa actccaattt gagtgcctga ctcttctaaaggcagatgtt 23760 tgttttagaa ttcagccttt ccagaccttt gcatgatgag gatggtgatgatttatagca 23820 caatggtgga aaaacagatg agtgcccctt aactcttaat ttgttttatgatttgaatct 23880 gtcttaatct ctaagataca gttcactttt taagtaggca gcttttctgtttaacatgat 23940 tttctaaagg attgctatag aagataatga aaagggacct tgagtaattgcacttttaaa 24000 tcacacaaga ttgttttgta tgtgctcaga tggtattttt tgaagttaatgttgtcattt 24060 cttttctttt tttttatttt cgggacaggg tcttcctctg ttgcccaggctggagtgctg 24120 tggtgtgatc ttggctcact gcagcctcca cctcctgggc tcaagtgatccacccacctc 24180 agcctcccaa gtagctgaga ctacaggtgt atgccaccac gcctggcaagttttttgtag 24240 aggcagggtt tcaccatgtt gccacgctgg tctcaaactc ctggactcaagatatctgtc 24300 caccgcagcc ttccaaagtg ctgtgataat aggcgtgagc caccgtgcctggcctaggca 24360 atttacttct ttgagtttta ttttcagtat atgagaaatg tggctaataacatttacttc 24420 attgcttgct ttgttattaa atatgatgat gcatgtaaaa tactcatgtttggtttatat 24480 gtgccaaaaa aataatacct gtaatatatt gtagagttag ggatgaaccactttactaaa 24540 tgtctccaaa taaccaattt ctaataattt agaatatgta gtatagctagtgtgcattgc 24600 attatgataa aagagtgctt attacctgtg cacctaattt ttggaagtcagattcatata 24660 gctttggtta atctttgttc tttttacatt ttattgtatt ttagacagggtctcgctgtg 24720 ttctgcaggc tgcagtgcaa tggcatgatc atagctcact gcagccttgaacccctgggc 24780 tcaagtgatc ctcccacttt agtgtcccaa gtattaaata gctggcattacagacatgtg 24840 ccaccatgcc tggctgtttc tcgttttttt tagagatggg atctcactatgttgccaagg 24900 ctggtctcga acttctggcc tcaaatgatc ttcttgcctt ggcccctcaaagtgctggat 24960 tacaggagtg agctactgtg tccagcctaa tcttcgttct tggagtcaagttgtgtaggc 25020 tttgtttttt gctttgcttt tttttttttc ccccacctct agtttttaatttaaaaaagg 25080 actggctttt agaaccactg gaaaatattt gttttggggg cagtgtctttagataacata 25140 aaattcagga atacaaattt tgggtggaag atagtacagc gtcattggttaagaatataa 25200 actctctggc cgggtgtggt ggctcaggcc tgtaatccta gcactctgggaggccgaggc 25260 aggcggatca caaggtcagg agattgagac catcgtggct aacacggtgaaaccctgtct 25320 ctactaaaaa tacaaaaaat tagccgggtg tggtggcggg cgcctgtagtcccagctagt 25380 cgagaggctg aggcaggaga atggcgtgaa cccgggaggc ggagcttgcattgagccgat 25440 cgcgccactg cactccagcc taggcgacag agcgagactc cgtctcaaaaaaaagaaaaa 25500 aagaagaata taaactctca aactgaattc taatcctggc actattacttactgccttgt 25560 gatgttaggc aagtaatgta accttctgtg atgcttaaat tttaatcttgtctgaaatga 25620 ggacaatgat aaaatgtacc tcctactttg gaaggattga atgaggtaatccatgtaaag 25680 catttagcat ggcctctact aagtgaccca cagcagttat tagtaatgacaaataagaga 25740 aaggagaagc agtgggcagg tgttcaagtg ctaacgtgta ttgtttagagcagtgttatc 25800 aataggagta taatgcaagc catgtgtaat tttaactttt ataatagctgtattaagtaa 25860 ataaaaagaa acaggtgttg aaattcatta tgataatgtt taatttaacccagcatatcc 25920 aaaacattgt tatttcaaca tgtaatgata tcaaaattat tgagatattttacatttttt 25980 aatactatat ctttgaaatc tggtgtatat ttatacctat ggcatgtctcaatttggata 26040 ctaaattctt tctgtctttc tgtccccact cctttccctc ccctcccctcccctcctttc 26100 ccctccttct cttccacttc cctttccctt tttccttcct tccttccttcctcccctccc 26160 tccctttaca cttccccctc cccatctctt tccccttccc ctttcccttccccttccctt 26220 ccttttcctt gtttctttgt ttttttttgc tgtttttttt ttttttttttttttccgttt 26280 tttgagacag agtcagccag gcacagtggc ttatgcttgt aataccagcactttgggaag 26340 ccgaggtggg tggatcactt gagcctagga gtttgagacc aggttgggcaacatggcgaa 26400 accccgtttc cactaaaaat ataaaagagt tagttggctg ggcgcggtggcccatgcctg 26460 taatcctagc actttgggag gccgaggtgg gcggatcacc tgaggtcaggagttttgaaa 26520 ccccctctct actaaaaaca caaaaattag ctgggtgtgg tagcaggcacctgtaatccc 26580 agttacttgg gaggctgagg caggagaatc acttgaactt gggaggcagaggttgcagtg 26640 agctgagatc aggccactgc actccagcct gggtgataag agcgagactgcatctcacaa 26700 aaaaaaaaga attagttagg caaggtggtg ggcacctgtg gtcccagctactccggtagc 26760 tgaggtggga ggattgcttg agcctgggag gtggagggag gttgcagtgagctgagattg 26820 caccactgca ccctagcctg ggccagggca aggccaccct gtctcaaaaggagaaaaaaa 26880 aaaaaaaaaa aaagaaatag ggtctcactc tcaccggctg gagtacagtggtgtgatcac 26940 agctcactgc aaccttgact ttctaggctc tagcgatcct cccacctcagtctcccaagt 27000 aattaggact acaagtgtgt gccagcacgc ttggctaatt ttttgtattttttgtaaaga 27060 caaggtttca ccatgttgcc caggctggtc tcaaactcct gggctcaagcgattctcccg 27120 ccttggcctc ccaaagtgtt cagataacag gcgtgagcca ccacactgggcactaaattt 27180 tcagcagaga tgcttgttct gtatttagat ttaataaaat ttacagtagaaaaagtagat 27240 tcgcctgggt gtggtggctc acgcctataa tcccaacact ttgggaggctgaggcaggaa 27300 gattgtttga gcccaggaat tcaaaaccag cctgggaaac atagtgagaccctgtctgta 27360 tactaaaaaa aaaaaaaaaa aaaaaaaaaa agaatactta aaaaaatgtattgttggctg 27420 ggcgtagtgg ctcacgccta taatcccagc actctgggag gccgaggcaggcagatcatg 27480 aggtcaagag attgagacca tcctggccaa tatagtgaaa ccccgcctctactaaaaata 27540 tacacacaca aaattagctg gacgtggtgg catgcacctg tagtcccagctactcgggag 27600 gctgaggcag gagaatcgct tgaacctggg aggtggaggt tgcagtgagctgagatcgtg 27660 ccactgcact tcagcctggc tacagagcga gactctgtct cacaaaaaataaaaaaaaaa 27720 gtattcttgt agttcctttt ttctccttgc aaggcatctt taatgtagaataagctaaat 27780 atagtaataa taataataat taaaaatgat gaagtttatt gagtactgctttgtatgtgt 27840 tatgttgtct tctaagcaca gatatctcat ttaatcttca tgatagcactatgagaatac 27900 tttaattttc cctgtttaca gatagggaaa atggggacca gagaaagttatagagcatag 27960 agttgaactc aattttgacc tccagagtct gtttttcttt tttttttttttatgagacgg 28020 agtctcattc tgtcgcccag gctggagtgc agtggcatga tctcggctcactacaacctc 28080 cacctcctgg gttcaagtga ttcttgtgcc tcagcctccc aagtagctgggattgcaggc 28140 acccgccacc ccacctggct aaattttgta ttttttggta gagatggggttttgccatgt 28200 tggccagggt gttctcggac tcctgacctc aagcggtttg cccgcctcagcctcccaaag 28260 tgctgggatt acaggtatga gccactgtgc ctggcccaga gcctgtttttctgacagtca 28320 caccgcttat tggttgctgg atagaatgaa aaagcaaatg gaagtacctctccccaccag 28380 tagtttttct gggtttcttt tatgggggag cgagggtaga gtaacatgtgggtagaggga 28440 cagtgtttca ggaacccatt tggatattta agaacaggac taaatacttaaattacttct 28500 gtgacaataa aaagataccc aggaagctac actctttttt agttctatattttttgcatc 28560 atacgtttaa aaattggttg ctcctaaatg aagctttaga atcctttgcaagatagtact 28620 actgcttcaa gcagctaacc accaatgaac aagaattatg agttcaaactctggcataga 28680 ccaaccccct aacttggatc tcctaactca taatccagag ttcttttccagagaactgct 28740 ccgcttttaa aaaccagtgc caggattcag atctcaaatg ataccattaaacactgatag 28800 gcaagaagca tttgtttcta tgagtaaata actctctgat gtattagaggctggtgttaa 28860 tttttgacta ggctgaactc tgagcgttta ttatttcttc aggtattaaaccttcagata 28920 atttcaaaaa cacagtttag gccaggcaca gtggctcacg cctttaatctgagtactatg 28980 ggaggcagag gtgggtggat aacaaggtca ggagttcaag aaaccccgtctctactaaaa 29040 atacaaaaaa tagctgggcg tggtggtggg ctcctgtaat cccagctactcgggcggctg 29100 aggcaggaga atcgtttgaa ccctggaggc ggaggttgca gtgagccaagatcacgccat 29160 tgcactccag cctgggtgac agggcaagac tctgtctcaa taaaaaacaaaaccaaacca 29220 cagtttatcc cttaggaaaa caccaaaaag cttcttatgt acatgaacttgtattagggt 29280 cacactttaa ggtggctcta gaaagtataa tgagagactt acagtaagactgcttttttt 29340 ttgacgtggc atctcacttt gttgtctagg ctggcttgga actcctgggttcaagccatc 29400 ctcttgcctc tgcctcccga gtagctttac aggaggatta caggtgtatgccactgtgct 29460 gggctgagac tgcagttttt aatgaatgaa atgccactgt gctgggctgagactgcagtt 29520 tttaatgaat gaaatgcctc tgtgcatgga ttgaagccag cttttatggcaaacattaac 29580 agtatggttt ggttccccat ctctgtgccc cttgagttat aacttaaattgttctgattg 29640 tagtattaac tgtgattagg tgttgatgaa agagatatgg agttctaattaagtaaaatg 29700 aagatctcaa ataaaattga agtagaatga aacgctatat gtaaatacaattctgctaaa 29760 caaatattta actgtggata ggcacagtga ctttataatg cagatagtcattttcatata 29820 tatggatata tataaatata cattatattc atatatatgg atatatataaatatacattg 29880 tattcatata tatggatata tataaatata cgttatattc atatatatggatatatataa 29940 atatacgtta tattcatata tatggatata tataatgtaa tacggtaattgatttttaga 30000 ctaaatatat aagttgaaac ataacatttt ctaatttttg gaaaattagtggtgttaatt 30060 ctggagacta gtaaaaataa atgattagag acgaatacac ttcatggtaacaaacatgct 30120 ggtgatacag ctttaaaact ttatggaaat ttacaacata caaaaataaagttcactgat 30180 gttttacata ttcatggcct agcatcacca gttgccaata ttttgccatcttggtattat 30240 ctgtttcact ctgctgcccc tcttctctcc tttctccctc accatgtagcattttaaagc 30300 aaattccaga tatcatttca cccatatgta tttcggtatg tatctctgacacacttgaac 30360 tttcctttca tagtacaact gtcataccat tgtcacaatg aataacattaacattaattc 30420 cttaatggca tctggtagca agaacatgtt cagattagcc tgtctctaaaatgtctttgc 30480 aatttgttcg tttacattag gacccaaata aagtcttcat attgcagttggaagatacgg 30540 ttctaaatgg tctcttaaat ctccaccctt ttcatgccct ttatttgatgaagaaactgt 30600 tatttaacct gtagaatttc ccacattctg tttggctgat tacatctttgtggtttggtt 30660 taacccctgt aatttcctgt aaactgaaca ttcgttctag agggtatttttttttggtga 30720 gcattgttta taagaggtgc tgtattcttt ctattagtta ccatcatgaggttggttcac 30780 tttcagtatt gctgggattg gtcagtggtt ttagattatg ttggcttgatctatccttta 30840 taaagttctt atcaactttc acacaaaggt tttcgcagca tctgaggattgttgactaga 30900 tccattatat aaggattgca aaacggtgat tttggaatta tatcactcctacatttatta 30960 gttgaaattc ttttgtaaag aaaactttta tttcatgtat ttactgtctttaaaatctag 31020 aagtggaggc tataaaaatt taattcctct cccacctcgt tttgccagtttttataataa 31080 tgagttggtg tcctaggaac ccactaaaac gaccagtaaa tttttcttctttcttttatt 31140 atttttagtg ttagggattt ttatgtatgt gatgtgtttt aatccattgtagttgctgtt 31200 tttcatgttt caattgtctc atctttggcc agttggaacc ccttcagatgggttcctgtg 31260 ttcttttgaa tattgtccca tcggttttca ataacttact ctctttctggcacaagggat 31320 atcaggttca ctttgtacat ttcctgctcc gcatctggaa tcagctatttctttaaggaa 31380 ccctggttgc tgagacatta atgacatcaa atatatatat aaaaagaaaagaacaagtag 31440 ccctggtttt ctcaagtggg aaatggtttt tagagatcac aatcagggtgttaagggtgc 31500 tcattgctgc tgggttggtc atgacttcta tggtttttta gtatttagatacaggaagta 31560 ctctctccct tccttccctc cctcctctcc ctccctccct ccctctttctctcccttcct 31620 cccttcctcc cttccttcct tccttccttc cttccttcct tccttccttccttccttcct 31680 cctttttttt ttttttaatt agagtaaatt atggctgggt gaggtggctcactcctgtaa 31740 cctaagcact ctgggaggct gaggtgggcg gatcacttga gcccaggagttcgagactag 31800 cctgggcaac atggcaaaac cctgtctcta caaaaagctc aaaaaaattagccaggcatg 31860 gtggtgtgtg cctatagtcc caactacttg ggaagatcac ccgagcctggaggtcaaggc 31920 tgcagtgagt tgagattgtg ccactgcacc ctagcctggg tgatagagtgagaccctgtc 31980 tcaaaaaaaa agagaagaat aaattttcaa ttcatactga tatttccagttttcatgaaa 32040 aattacagag gttttgttta acttatttta tgatttattt atttattttatggtgaaaaa 32100 tatgtctatt tagtgttagc cagctgggct cactttagat gatcccaattttgttggcaa 32160 cgtcatcata gtcaggaacc ggtagaacat gggccttctt tccatcaggcctgatcaggg 32220 tgttgatatt ggccatgtca gtgccacaga gctttttctt agcctgtttgatctggtatt 32280 tgttggcctt gacatccaca gtgagcacaa gaatgttgtt gactttctgttcttttcatg 32340 gctgactcag tggtcagggg aaacttgata gcatagtggt caagctggtttctcctggac 32400 tggaccagtc ttccaaggat atttgggctg cctccagaac agcagcagtgttttgggctg 32460 tccttaggtg ggtgacgtga agatcttctc ttttttgtgt ggctgtggatacggatacct 32520 ttcagcactg ctgttttggc ctttagagcc tttggtttgg ctttgacgttgggagagcag 32580 gggcttccct ctttgcctgt gatgccttct tgatgagtac agccatgtaacttcttttat 32640 agttgtgtct tttttttttt ttgggacgta gtctcgctct gttgtccaggctggagtgca 32700 gtggcgtgat cttggctcac cgcaacctcc acctcccagg ttcaagcgattcccctgcct 32760 cagcctcccg agtagctggg attataggca ccctccacca tgcctggctaatttttgtat 32820 ttttagtaga gtcagggttt cactatattg gccaggctga tctcgaactcctgacctccg 32880 gatccgcctg ccttggcctc ccaaagtgct gggattacag gcgtgagcctccgtgcctgg 32940 cctttttttt ttttaaattg agatggagtc ttgctttgtc cccccgcaggctagagtgca 33000 gtggcatgat ctcggctcac tacaacctct gcctcccagg ttcaagcaattctcctgcct 33060 tagcatccca agtagctggg attacaggca cgtgccacca tgcctggctaatttttgtat 33120 tattattaga gaccaggttt tgccatgttg gccaggccgg tctcgaactctggacctcaa 33180 gtgatctacc tgccttagcc tcccaaagtg gtgggattac aggcgtgagccactatgccc 33240 ggcctatatc attttttatc ttatgctaaa aatgttggtt cctaacaacattaacattat 33300 attatacata taacacatat tagctttgag ataacaatac cataatgtagtttgagattc 33360 ctttgtctgt ctatttacat ccttaggctg tattccagta gggatgtaagctcagaatac 33420 tttttaaatg aaaataaaat tttataaatt ttaataaaaa ataatattttaaataaatat 33480 taatatttaa gatacttgaa ataataattc tctgtgtatt tatgtcagcagttgaaaata 33540 gaacatttac ttcagtttgg ttttggtttc taaggagtgc tgttttttccttttcaattt 33600 ttttgatgta aaatatttac atggtttcaa aatgttgagc atatttttaaaaagatatgg 33660 ctgggcacgg tggcttgcgc ctgtaacccc agcaatttgg gaggctgaggcgggcggata 33720 acttgaggtc aggagttcaa gaccagcctg gccaacatgg tgaaaccccgtctctactaa 33780 aaatacaaaa aatcagccgg gtgtggtagc aagcgcctgt aatcccagctgctcaggagg 33840 cttgaacccc tgggttcaag cgggagaatt gcttgaaccc ctgggttcaagcgggagaat 33900 tgcttgaacc cctgggttca agcgggagaa ttgcttgaac ccgggaggcggaggttgcag 33960 tgagccgaga tcacacctct gcactccagc ctgggtgaca gagtaagactctgtctcaaa 34020 aaaaaaaaaa aaaaaatgct gctggtttgc atatcattat tctgcccatatcctatcagt 34080 aacgaaagag gtattatttt actgtatgtt tttacttaaa atttaatttatgcttacatt 34140 aatttttaaa ggttcagatt tgaaagtcca gtgtcaattt tccaggtggccaggtggtac 34200 tagatgtcag taaatagttc ttgtttatat attgcgtttt ataattttaaatattttact 34260 gggttttgga catattgtat gtcaatagaa ggcaactgga actctaattttagaaactaa 34320 gattatgata tttgtagatg atcagtttta ctccttgtag tccaccccctccaattactt 34380 aataactatt ttaagatgtt ttacagtcta atatactgaa atttctgacataaagtcaga 34440 cttccagatt acattgctta aatttgccaa actaggcatt tttctggtggaggagaaagg 34500 aatcttttcc taaaagtctg gtatatatat attttttgct taatgcctagttaccatgga 34560 aatagatgtt agaatcagct cttaagtagg taggtagtga caaactggctgtattgctga 34620 attgagttgc tagactgtat ttggcccttt cctgctttca tctttccaccttctctttca 34680 tcattttttt tttttggttt atgtgctttt tagaatattc tatatagttaaatccaaact 34740 tgacttaacc taggtttgag caacaatttg cttttgctta caatttatactatctatagg 34800 tagacatttt acctcatatt tacatatgta tagtataagt agataattgtactgacttgg 34860 cacatcagct gttaatgctt ttaagaaaag gtctggaaag acctgtaacaagttaatagt 34920 gatggtctct agctggtgac agtatagaaa atgtttaatg tctactttgtgaagttacaa 34980 tgtttggttt ttaaaatgag catgtactac ttcattatat aaaatttttttacttgtaga 35040 agtttgtgtt aatgatcagt atttaaaata tagtaaaaaa taagagaggaaaagtggaaa 35100 tcctctagta gcatatattt ttttggcctt aactttgacg tttgtgtgatgttgatcttt 35160 agttactgcg cttcggtact gaggatagca gcacctatgt catagtgttgctgagatgaa 35220 atgagattac taaagcataa tgcttggcac aaggggagca gttaataaaataggagctct 35280 ggtttttgtg gatcttagat ttgctgccct gtaggttttg ggaaggagtagtctggctct 35340 acaggaatga ggaatgtatt gtggagtctt agaagagcta tctacgtattcatttgaatc 35400 cacaaccttc cctcggagct ttcttcttga ctatgtagtt ctactttagcttttctcttc 35460 tgtcagtata tttctagtca tttccagttt tctgctcagc agagactttggggtccatgt 35520 tgtacctgtt ctgttactgt tacataaaag tagatctata aatttatcttgttagtaatc 35580 aaaaaagtta aagttcaagt tttatgcata ttctggatga gttttaccttacccaacctt 35640 taatttggtc atgtgattga aaaaaagatt ttttaggggt tccttttcctatcactgtag 35700 atggaaaatt tggaattttt tttttttttt ttgagacaca gtctcaccctgtccctcagg 35760 ctggagtgca gtggcatgat ctcaactcac tgcagcctct gcctcccgggttcaagcgtt 35820 cctcctgcct cagcctccca agtagctggg attacaggca tgagccaccacaactggcta 35880 cttttgttgt atttttagta gagatggggt ttcaccatgt tggacaggctgttcttgaac 35940 acctgacctc aaatgatcca cctgccttgg cctcccaaag tgctgggattacaggcgtga 36000 gccactgcac ccagccaaaa cttggaattt ctaagagcct ttattaatttgtaaatcagt 36060 aacattggaa ttgaatattt gaaactggaa ctgtccccag aatattgaggatgcaaagtt 36120 tctgtaggca caattcttca caaaggcaga ctcttgggga caatcaggatgattgcaaaa 36180 tcatatgaaa tataatatgt aacgaaatga agaagcaaac aacttacattataagttgta 36240 tctgtattat taaagtccaa atatcctagt ttaatttttt gaatattcaaacatatggct 36300 gaaaagtttt aaaaaatact tataaaagaa tatagtaaaa accctttcatcctacctttt 36360 acctccaatt gttaaatttc ccttctttct gtacagtcat tgtttttgtgtattgtttcc 36420 acacgtagaa aggcaaacac aagtttgccg tcttacttcc ctatctctcctaccctttac 36480 tttcagcttg taaattacac acactgttct gcacttttaa attttgtcacttaacacatc 36540 tgggagatgt ttgtgttatt attacataga gatcccctca tttgtgggtgtttttttgtt 36600 tgttttgttt ttgttttttt aagacagtct tgctatgtca cccgggctattttttagtat 36660 tttttgtatt ttttagtaga gacggggttt ggccacgttg gccaggccagtctcaaattc 36720 ctggcctcaa gtgatctgcc cacctcggcc tcccaaagtg ctggaattaacaggtatgag 36780 ccaccacgcc tagccttgtg tgttcttatt aacaactttt attgaggtataacttgcaca 36840 ccaaaaaaat taaaccattc taagtgtaaa aatcaaggaa ttttagattcatttttaatt 36900 aaaaatttat tttatttatt tatttattta ttattttttt gagacagagtcttgctctgt 36960 tgcccaggct ggagtgcagt ggtgcgatct cagctcaccg caacctctgcctcccaagtt 37020 cacgccattc tcctgcctca tccttccgag gtagctggga ctacaggcacccgccaccat 37080 gcccagctaa tttattttta atttttttta tttttagtag agacggggtttcaacgtgtt 37140 agccagggtg gtcttgatct cctgacctgg tgatctgccc acctccgcatcccaaagtgc 37200 tggaattata ggcgtgagcc accacgcctg gcctattttc ttcttttagaaacaaattgt 37260 tactctgtca cacaggcagg agtgcagtag cactatcata gttcagtgttaacctcaaac 37320 tcctgggctc aagcaatctt ccaacctcag cctcctgaac aggtgggactgcaggtgtgt 37380 gctaccatgc ccagctaatt aaaacaaatt tgtttgtaca gacacggtagcactatgttg 37440 cctaggctgt tcttgaacac ctcctctcaa gagatactgc cacttggcctcctgaagctc 37500 tgggattaca ggcgtaagcc tccacacctg gctgttatgg aatttttgtaaatgtctagg 37560 gttgttcacc catcaccaca gtccagttta agaatatttt tgtaatccttaaaaagttgt 37620 ctccccgtct ctggtctttc atgcccatgt ccagctgcaa acaaccagtttcctcaattg 37680 tttttgcagt tctgtatagg acagatgtat aagatttacg atgtaacataatttatttag 37740 ccacacccct atttttggac atttaggttg tttttgacct tttgcttttacaaacaggtc 37800 tgagatgagg taactttgta catatattat ttcatacatc tgtaagagtatctgtaggat 37860 taattcctaa gagtagggtt actgattaaa gcacatgtgc tttttttttttttttggtga 37920 tggaatctca ttctgtcgtc caggttggcg tcatcttggc tcactgcaacctctgcctcc 37980 ctggttcaag cagttctcct gcctcagcct ctggagtagc tgggattacaggcatgcacc 38040 accatgcccc gctaagtttt gtatttttag tggagacagg tgtcaccatgttggccaagt 38100 tggtcttgaa ctcctgacct caagtgatct gcccggcctg cattttttatttctataatc 38160 gtttcccgtt caggttatac agatttacat tctcagcagc aatatgaaggctaacatttt 38220 cccataactt acctgccgtg tacattacta gacttctgaa tttctgaatttttgccaatc 38280 tgatagtgaa aaatggtgtg ggtttttttt ttttagagat agagtctcactatatcgtcc 38340 aggctggagg gagtgtagtg gcatgatagg ggcacactgc aacctctgtctcccaggttc 38400 aagcagttct tctgcctcag cctcccaagt agttgaaact atggatgcgtgcgaaaaatt 38460 gtgttttgaa ttgtttttat ttagtaggtt ttacatcgaa tttatgaatgagtatgagca 38520 tcttttcaag tgttgagtcc attgtaattc tttttatatg aaatatccttttatgtcata 38580 tgcccatttt tctgttgaat ggttggcttt tttttttttt tttgaggcagaatctccttc 38640 ctgtccccca ggctggagtg cagtggtcca ataacggctt actgcagccttgaacctccc 38700 aggctcaagt gatcctccca cctcaacctc ctgagtacct ggggctacaggcacatgcta 38760 ccatgctggc taatttttaa attttttaat agagataagg cctcactatgttgtccaggc 38820 tggtctcgaa ctcctgggct caagcacctc acctacctcg gcctcccagtgttgggacta 38880 taggagtgaa ccactttgcc tggcctgaat ggttggtctc taaaaaaaattactgatttt 38940 agatgttctt tatgtatgag agtcaagctc tttagtgatg tgaattataaataacttttt 39000 gtagagtttg tcatttatat tttgatatat ttgctttgtg tatttttttgccattgagca 39060 attttttaat ggttaaattt atcaatattt tatagctccc aaactttgagttaatttact 39120 gtttgaagta agtcatacat tgaattccct tccaggatca taatttattatgtcactttg 39180 aatttagctg gacttttggc cagacctact gacttgcatc tgtgatgaataactttgaat 39240 atatgttatc tcacacatgt gcaagtatag ctattggatg aatttggccatttattcctg 39300 gggccagggt tgtgtgtttt taattttggt aattattgtt gaataaatggatctttcaag 39360 actgttaaga tattgcattg ctaataactg gtgcagaaat taatggtgatttctgttcaa 39420 gatgccagat tgttaaactc ctctaggatt agaaaagttt tggttgaaaatagaacactt 39480 aactgtaact aagagtcttc ttttttcatt atgtgttggc ctttaaggcaccagaattat 39540 acttcagaaa ttccttggga gaatttgggt tatgtcttaa aataaaaaatgttcatgttc 39600 tttgtgctcc cttttgatac ctacccagaa gcagcccatg tacaggtgcacaaggaagct 39660 ggcatgaata tgtttgtggc tgccctcttt aatagggaaa aacagaaaaccacttaatat 39720 caccagtaga ggacaagcta aataaccatg gcaaattcct acaatggttatagcacaaaa 39780 ttaattcgac agtttgtgtt tactgatgta gaaagatctc taaaacattttttgaatgaa 39840 agaaaagttg aaagacagta tattaaatgt agtaccagtt atgtaaaaatagattcacag 39900 aaatgttatt aaatatggat acatgtacgt gtatgtaagt gaaggtctggaaaaaacctg 39960 gaagaacata tgccaaaatg agaatagtga ttattaatgg gaattgagtgtagtaattta 40020 ggacttactt tttttttttt gaaatggagt ctcactctgt ttttcaggctggagtgcagt 40080 ggtgcaatct cagcttactg caatctccgt ctcctgagtt caagcagttctgcctcagcc 40140 tcctgagtag ctgtgactac aggtgcccac caccacgcct ggctaatttttatatttctt 40200 taatagagaa ggggtttcgc catgttggcc agcctggtct tgagaaattcctgacttcag 40260 gtgatccacc cgccttggcc tcccaaagtg ctgggattac aggcatgagccactgcgccc 40320 agccacatta atgttttatt atttttacaa ggataatatg ctcatagactgtacaactaa 40380 aggccaattt aaaatttatt tggaaaaaat acattgtttt gtacataggtccctgtaaag 40440 gcagagtaag actggaaaaa taccctaaat aaggagacag tagttatgtccccgagggat 40500 gtgggaacag agaagataaa aggagacatt tgctttttgt atacttctgtatttttatag 40560 ttttataaag caaatgaatt catgcattaa tttgtatgct gaagaaaggactaaattctt 40620 taggaccagg aattcatgta agagtatctc attttgtcaa gcttatttaacagtacttag 40680 tgaaaatagt agctgccggt tgaattactg ggtagagaat aaaggtgtcaacagaaatag 40740 tttactaatt tgctgataat taggcattaa tttgcttgat gtgatgcatgtttattactt 40800 tagggcaagt agaaaaataa aaataagtag attctttctc atataccatcatgaacactg 40860 gtagtttctg gtacattcaa gacagaacac acaatgagac tgttcttgccctgtagcctc 40920 acctggaatt tccttttgtc ttctgctttg ttactacaac ctcccctgcagtttgggtgt 40980 ctctctagtt ctcttttctt tgtttagtag tctgggatca gatctgctcttctgaaatag 41040 aagtcagcag tgaggagaaa aaaggtaaaa aaaaaaaatt tattttttctgagacagagt 41100 cttactctgt cgtccaggct ggagtgcagt ggcacgatct tggctcactccagcctccgc 41160 ctcttgggtt caagcaattc tcctccctca gcctcctgag tgtctgggactacaggcacc 41220 tgccaccacg cccggctaat tttgcatttt tagtagagac agggtttcaccgtgttagcc 41280 aggctggtct ctaactcctg acctcaaatg attcacccac ctcaccctcccaaagtgctg 41340 ggattacagg cgtgagccac agtgcctggc caaaaggtaa aatttttaatccctttattc 41400 agttagtcac taaatgtgag aatgtcttta cccagttctt ctctctccatcccagcatta 41460 gtgcaagcca ttgtatctca ttctagatta tggtagcaga ttcctaactgatctgtctga 41520 ttcagccttg tatttgtaca ttggtatctg tttttcacat tgcagccacagtgatctaag 41580 acatcagata atgtaactct tgctcataaa actttgtagt agttcttctcctgttgctat 41640 caggccttgc ttctccagaa ctctggctat ggtcattaat aattgcagctttctatattg 41700 tctactttcc tcatgcacta tagctcttct ccagaatcta tattgtttctcatttcaacc 41760 catcagaatt cttttactag attaccagtc atccttcagg tatccttctctgagatccta 41820 agatagattt acgtccttct tcgtgctcct cttgtgcaga atctgaccatagtagttaac 41880 ttcatatggt aacttaactg tttaacagat tcctccacta cactccatgaaaacaaagac 41940 tgagttttaa atcaagtctc tggttcctaa ccagcacctg atacattataggctttagtt 42000 taaatgtatg aataagcctg ggtgcgatgg ctcatgcctg taatcccagcactttgggag 42060 gccaaggtgg gaggatcact tgagtccagg agtttgagac cagcctgggtaacatagtga 42120 gaccctgtct ctacaaatta tcaaaaatta gctgggcatt gtggtacatgcctgtagttc 42180 tagctacttg ggaggctgaa acaggctaat tgcttgagcc caggaagtcaaggctgcagt 42240 gagccacaga gctcagcttg aataacagag ggagacccta tctcaaaacaaaacaaaaca 42300 aaacccaaaa gccagaaaca aatgtgtgaa tgagcgttaa ctcagtcattttttctctca 42360 tatcattttt ttctcttgtt ctttccctac ctagtaatta ataggttgtggctcagtttg 42420 aaaaacactt gtgtaaggaa tcctgattta ctagttagaa ctctcagaatgagaactctc 42480 tgccagatct ctaaatattt agtaattttt tattctgttg ttaatggtattttaaaaatt 42540 caatttctga ttgttgctaa tgtataggaa tcctattgac ttctctatattgtgtatcct 42600 gtgatcttgc taaaaccacc tattggttac agcaggattt ttgtagattcctttggattt 42660 ccttggtagc ggatcatgtt gtcagtaaag gcagctacta ttcttcatgtccaattcaaa 42720 tatcttttct ttcttgattg cattggctct ccaaaacaat gcagaatagaagtggtgagg 42780 tggatatccc tgtcttgttc ttaattgtag ggaaaagcat tcagtctttcaccgttaagt 42840 ataatgttag ttgcagattt ttcatggatg ccttttgtcc gattgaggaagtttcattct 42900 gttcctagtt tgctgagaga ttacatcagg agtggcttat ggattttgtattaataaaat 42960 gcattttctg gatatattga ggtaatctgt ttttaaagtt tgttgatacgttaattacac 43020 tcattgactt ctgaatttta gaacaatcct ccatttctac aataaatgcacttggtcatg 43080 atatattacc ttttagaaca tattgttgga ttctacatgc tagaattttatttagaattt 43140 ttgggctggg cgtgatggct cacacctcta atcccagcac tttgggttggcttaagtcca 43200 ggagtctaag actagcctag gcaacatgac aaaaccctgt ctctgcagaaaataaatttt 43260 aaaaaaagtc agctgggttt ggtggtctgc acctgtaggt cccagctacttgggaggctt 43320 gggaggctga ggtgggagga tcacttgact tcaggagttg gaggttgcagtgagccaaga 43380 tggcaccact gaactgcagc ctgggtgata gaacaagacc ctgtctccaaaaaaaaaata 43440 tatatatatt gtttgcattt ttgctcatga gggatattgg tctgtagagatgtggttttg 43500 ctatgttgcc caggctggtc ttgagctcct ggcctcaagc agtcctctcctctcagcctc 43560 ccaaagtgtt gggattatag gcatgagcca ccatgtccag cctctagttttattttctca 43620 taatgtcatt atatatcaga ataatgctgg ctttgtagaa tgagttgggaataattccaa 43680 attttaattt tagagatggt atcttgttat gttgcatagg ctgttctggaactcatgggc 43740 ccaagggatc ctctcacgtt agctttccaa gtagctggga ttatagccatgagccactgt 43800 gcccagcaat ttttatttga aatactaggt cataatcaca ttatggttttaaagatcact 43860 tgtaatagaa aaagctagtg aagtatatgt aattaaattt ctctgatcaattcatcacat 43920 tgcatcatca attttatagg atacttggag agcagataat ttaggccttgaagaattttt 43980 tttcccccag agacggagtc tcgctctgtc gctgaggctg gagtgcagtggcataatctt 44040 ggctcactgc agcatctgcc tcccaggttc aagcgattct tctgcctcagcctcccgaat 44100 agctgagatt acaggtgcct gccaccacgt ccagctaatt tttgtatgtttagtagagat 44160 ggggtttcac atgttggcca ggctggtgtc gaactccgac cttcagtgatctgcctgtct 44220 aggccttcca aagtggtggg attacaggcg tgagccactg tgccggcctggccttgaaga 44280 attaatgttg atttgtttta aatgtagaat gaggctgggc gtggtggttcagacctgtaa 44340 tcctaacact ttgggaggcc gaggcaggcg gatcacttga ggccgggagtttgagaccag 44400 tctggccaac atatcgaaac cccgtctcta ctaaaataaa aaaaaaattagcagggtgtg 44460 gtggcgcatg tctgtaatcc cagctactcg ggaggctgag gcatgagaatcgcttgaact 44520 gggaggcgga ggttgcagtg agccgcgatt gcaccattgc actccagctggagcaagact 44580 gtcttggaga gcgagactgt ctcaataaat aagtaaataa aggattggtttcattttctt 44640 tagggtctaa gttaactttt tcttaaaagt atcagcaaga aatcttaaaattgactgtct 44700 taagtaaatg ttaaataata gtacttaata ttaataattt tataatttaataatgaacat 44760 aaagcatttt atttgatttt aacattttct ggttattaaa aaactcatggtagaaagtat 44820 ggaaaatcca gaaaatcagg taggaaaaag atcactcgta attcagacgttacagaggta 44880 actactatta acattttagt atatactttc aaatctttac ctgtgtatttcttaaaacat 44940 agtttgtaat catctttttt caacatttta aacattagct tctgaattagtaggtaagtt 45000 tatgttggtt acagataggt ttttttaaga gggtgttaag ttgttactacctgataaggc 45060 agttatggta tgacatctgg atttaagctc tttttcctgc ctctagtgaattatgtcctt 45120 aaaatcactt aaagctccac ctctctttgc tcttttatac cagtgggtaataatactttt 45180 ttatttatct acataattgt ttttgtgatg aaaatgaatg tgttttaaaagatacataaa 45240 acttcttatt ttgttataaa atgaagtagt atcggccagg tgcggtggctcacgcctgta 45300 atcccagcac tttgggaggc caaggcaggt ggatcacaat aggagtcgagaccatcctgg 45360 ctaacacggt gaaaccccgt ctctactaaa aatacaaaaa attagccaggtgtggtggca 45420 ggcgcctgta gtcccaacta ctcgggaggc tgaggcagga gaatggcgtgaacccggtag 45480 gcggaggttg cagtgagccg agatcgtgcc actgcactcc agcctgggtgacagagactc 45540 cgtctcaaaa aaataaataa ataaataaat aaaattaaat agtatgtaatgtaaatattt 45600 ccatgtctgg ttaaatttgg ttgtctttaa gtattcctgt tttctctttctctaattttt 45660 ctgaaaattc agggtacaat aggagttctt aaactttttt gggtctgtcgaaagtacagg 45720 ctttattgtg tagatatatc atctctgaaa attcttgtag ggtcttcagaaatccacaga 45780 aacagtgtta tggtttccag gttaaaattt tccatattta taatagttttcacccattgt 45840 ccatggcacc ttggtttacc tacttgtgct tccaaaagtt tgacacactcatacaactaa 45900 ttttcgcatt cactaaattt gcattgactg ttatcccaaa gcctaccaaaaagaaggttg 45960 ctttgcgttg attatatcct gtgaagtagc cttgagcagc aaatattactttttgtaagt 46020 tagagacagc gctgaggaat aaaatcaatg catttctcaa gtagatcttgggtttctgag 46080 accatgtaaa aactttgaat cctgaggata taattaaaca aaactgtaaacagttttcat 46140 aagtatcaca gggctaaggg atgagcaagt gatactaggt taaccaggactccctgaaga 46200 cattgttaga ttatatgctt taggagtgat tgtgtttttg atgattttcaattccttcaa 46260 atatgccact atattctagt atttcactgt ttttatttca tagcagagtccccttctgaa 46320 tcatggtgta tattcatgat gtatgttcct aggctagtat tttacccaaaataggaaagt 46380 tttagagatc attttgtatc tgaaaagtat tcccttattt ataatgttgatatctaatat 46440 aagtaatgtg ataatttctt ttaatttttg tatttctgat tttttcagcatttcttattg 46500 gtcttctgaa ttgtataaag aacaggtttt taaaaataca ttcaccagcctccctaagaa 46560 ttgcatgaga gtacctctta ccactgttaa ataaataact cctgctccctttgttttttg 46620 tttttgtttt tgtttttctg aggcaggctg ttggtctgtc actcaggctggagtgcagag 46680 gcatgataac aactcactgc aggcttgatc tcctgggctc aaatgatccttctacctcag 46740 cctccaccac gagtagctgg ggccacaggc atgggctacc acgctcagctgtttttttga 46800 gacggaatgt cgctctgttg cccaggctgg agtgcagtgg catgatcttggctcactgca 46860 acctctgcct cttgggttca atcgattctt gtgcctcagc ctcccgtgtagctgggacta 46920 caagcatgca ccaccacgcc cagctgattt ttgcattttt agtagagacagggtttcacc 46980 acgttggcca ggctagtctt gaactcctta cctcaggtga gccacccgcctcagcctccc 47040 aaagtgctga gattacaggt atgagccact gtgcccggcc tttatttttgtttttaacag 47100 aggcagtttc cctatgttgc ccaggctggt tttgaactgg gctcaagtgatccacccgcc 47160 ttggcctccc aaagtgctga gattataggc tgagccatcg cacccaaccctcctgctcac 47220 tttaaagcat gctgtataga gttgtcctgg tgagaagcca ccttgatggcaagcttgtaa 47280 gctgtagagt tggtgagtgt ttacttagga ctcacatttg aaaccgcttttttttttctg 47340 ttatctttta atatgtaaaa taataatcag aagttactca ggactctttttttttcgttt 47400 ttatacatat gatttaattt ttgaactgag aattagcgct cacccaatattaagaatttc 47460 tttcaaaaaa cgtgctcggg tcaacacaag gcctctctgt acattatccttccgaaacca 47520 tggtttcttt gcttgcacct ttatccccac tgccttgcca gtccttcccgccagcaggac 47580 tggaagcctg gcgactccct aggtcacagg gttttccttg ccataaccaagtgggctctg 47640 cgcgtggctg atggcagccc caccctgcgc ccctgctgtt aggcaccgagcaaggagggg 47700 cccctggctg ttcttcgcct gggcactggt gtgctttcgg cgatcctggccacctgccca 47760 cctgaagctg ccatcttggg ttctggcagg agccgtttgc gtggcgaggagcggagaggc 47820 aggaacccag tgagctgctc aggacctgag ttgtgggaga tcatgactatttctttgcga 47880 tgcttcccca atattcttta agtcaatttt gtttgtcaaa tagcctgattttagtgatcg 47940 tctcttgtta gagcctgccc agtatgtatt ccaccttgtt tgttaacaacataccaattt 48000 cccttggatt gtcagtgaag gtgtcctgtt ctttactgta caagaactaagcatgtgatt 48060 caaataaggt tactggatat gttaaaaaga ataagacaat ggaaataaaataatattaaa 48120 atgttttatt ttattaatgg gtggagtaag agtaatacaa atgttttagacttgaagtta 48180 ctgtcagtgt ttttatcatt atatgactgt caaagagctc ataatagaattaagagaatg 48240 tgttggatat ttccagcacc agtgttagat ttggtaattc acaaggtgcgctcacaggac 48300 tcagtgtatt gtattagtca tgtttacggc tgcgatttat tacaagaggataggaagcat 48360 tatcagcaaa aggaaaagat acattgggta aagtctggag gaaaccaggtgcaaatttct 48420 cccggtagag ttacacaaga cacacataat tcccctaacc aagaattgtgacaacatgaa 48480 atgttacaac ccgggaagct ccttaaaact cagcatccag ggtttttagtgggagctgtt 48540 cacataggta ctgcatgtct ggcatatatc aaattctaga ctctgataatgaaagcttaa 48600 gccatattgt tttagacaca accacccact cttaccagtt ggtggtaggaaccctcccta 48660 aatccaggtt cctgtgagtt cttagccaag ggccagcttt gcaagcagccctttctaagg 48720 aaagccatct caggtctgct gttaactttt ttctgcatag aagattagctttttatttac 48780 taattcttta gttgtttgcg ttgattattt tatctcaatt gctgatttctttgctagagt 48840 cctctcaatt tttaaataat gtattatttt aaattggtct gcctttatggccgtatttta 48900 ttttgtgtgt atgttacatt gttttcatta tcactatcct gatatctttcaggtctttaa 48960 tgacttctga aatttccaaa atggctttca gattttgatt cctaccagaatacctcatac 49020 tacgtggttc aggaaactaa atggagattc tgttagtttc tgagcatatctattacaaat 49080 atctacttaa gacctagagg aataacctct gggtcagtgt ttctcagtcttgccgggggt 49140 ggggggtgca tcagagtgtc ctggagggct tgttaagaca caaaatgctgtttccctccc 49200 ggagagtttc taattcggaa tgtctggagc ggtgcctggg aatttgcatttctaacaaat 49260 ttccaggtgt tggcttggcg ctgtatgcac tttgctgtaa tcccagcactttgggaggct 49320 gaggtgggtg gaccacttca gcctgggagg tggaggctgc agtgagttaggatttccagc 49380 ctgggtgaca gagtgagtga gaccctgtct caaaaaaaca aattttcagatgttgctgat 49440 ggtgttggtc ctgggagcat actttgggaa ccacttttct agtttataatacaatccaca 49500 aacttatgta aacaagaaaa atgtaatctg taaaattccc atgtacaggaataccaaata 49560 cagtgtgcca aaagtggtag gacaattgaa gacttcattg tcaaccctgctgtgttgtgc 49620 agctgccact tttccctcag aatgtgtgtg ttcacaaggt gtgttgcttttgtagatcaa 49680 cataagaaat acttactagt aaagtttatt tcttatctgt ttttaagataagtaaattaa 49740 cagttcacta ttttttgtgt gtgctcagtt aatcttcctc atatcacctggaatttatca 49800 tctcccttgg gtaatcacag tctagtagat tctctcttca ccattttcttagggaaaatg 49860 gattggcaag ggaaatgcta gactatttta agggatttat tggcttttctgtcccactgg 49920 gaatgcttat aggaggggat gccatgaaat gtttttcatg atctcagtggggatcagaaa 49980 attccagcag aacggaggct aggtgggcat ggttaacaca tttaggtaggagggcatgtt 50040 tgataatgaa attctgaaaa tgtttgcatg tgcattgaga tccctaggtagataagtaac 50100 ctagtaactt atttgattgg tatagttttt taaaactcca ggtctgatgataatctaata 50160 taaactattc tattgaatgc tttgaaattg agtctgttta gttctgagaggccattgaaa 50220 gaaggcaaga ccgagtatcc tggaaggcct gttacatacc cctgtatgtagcatggccct 50280 ttctacttgc ctttgctaat gatactttgt gtgtgtacat ttacttaaatagctgtgcag 50340 tgggataggg gaataatact caggttttaa aatctgattt tggctctgacttaaccatta 50400 attactggtg actcaaaaga ataccactgt gacctaccag taagagtaggtgcatatgga 50460 gagtcaggaa atagcatttt gatagagttt tgacttaaat atgtttccttacactcatct 50520 tgaaattgtt tcttatggtc ctaagtgaat agttggaata gatatcctcagcaactaata 50580 gaatctccat ttagtgtgag gtagtatgga aggaatggcc aagaaaagccatttagtgcc 50640 accattaaaa tcctgaaaga tgctgggcaa ggtcatcagg agaaaaatgtaaaacaaaac 50700 caatgaaaca cacacacaca cacacacaca cacacacaca cacacacacacaatataagg 50760 cattaaaaat ggtctcattt acattattat tattattttt ttttttttttgagacggagt 50820 ctcgctgtcg cccaggctgg agtgcagtgg cgggatctcg gctcactgcaagctccgcct 50880 cccgggttca cgccattctc ctgcctcagc ctcccgagta gctgggattacaggcgtgcg 50940 ccaccatgcc aggctaattt tgtattttta gtagagaccg ggtttcactatgttggtcag 51000 gctggtcttg aacttctgac ctcaggtgat ctacccttct cagcctcccatagtgctggg 51060 attacactgc acccagcctc taaaatcttt tttaaatttt agagagagagtctctctctg 51120 ccacccaaac tgaagtgcag tggcacgatc atgtcttgtt gcacctttggcctcctgagc 51180 tcaagtgatc ttctcacctc agccccctga atggttggga ccataggcgtgtgccactga 51240 gcccagataa tttttttatt ttttatagag ccatgtcttc ctttgttgctcagggttttt 51300 cttggcaaaa gcatattgcc agaacatatc tttttttttt tttttcggagatggtgtttt 51360 gctccatcac ctaggcttga ttgcagtgca caatctcgac tcactgcaatctccacctcc 51420 cgggttcaag cgattctcct gcctcagcct cccgagtagc tgggattacaggcacccgct 51480 accactccca gctaattttt tgtattttta gtagagatga ggtttcaccatgttggccag 51540 gctggtctca aactcctgac ttcaggtgat cccccctgct tcggcctcccaaagtgctgg 51600 gattataggc atgagccacc gcgcctggcc aacatctctt ttttaagagaaataacttgt 51660 aatttctttc tttccttttt taaagagaca gggtcttgct gtgtctcccagactggagtg 51720 cagtggtgtg atcatagctc aatacagcct tgaacttctg ggctcaagcaatcctactgc 51780 ctcagcctca caagtagcta ggactacaga tgtgcaccac catgacaattagtattcttg 51840 ctttattttt tctttttatt tgataagaag tttataactt tgaaaaatatgagtagaaat 51900 gtaagagtct gttgaagatg taaactattg ttttttaaaa agtgtaaatatatttttaat 51960 aagattgtag ggccagacgc atctggtaag ggcttaaaag ctgatttttaaaaattgata 52020 gccagaggtt atatcttact aatgtttgct aatgctacta attagcgttagtttgctaat 52080 gctactaatg ttataataat gatctgatga cctcaaatag cttttcaaaaaaaaaatctt 52140 tttttttttt tttttgcttc gtatgttcac atttcattat ttcttactggtcaagatttt 52200 ttttcttttt ttgaaagtct ctgtctgtca cctaggctgg agtgcaatggtgtgatcaca 52260 gcttgctgca gcctctactc cccaggctca agtgatcctt ctgcctcagccccctcacta 52320 gctaggagcc acaggtgtgt gctaccacgc ctggctaagt ttttgatattttgtagagat 52380 gaggttttgc cgtgttgtcc cggctggtct tgaactcctg ggctcaagcagtcctcccac 52440 cttggccttc cagcgtgttg ggattacagg cgtgagccac tgtgcccagcccaggatttt 52500 tgtatatttt gacagtttag cttaaaaatg taaactggaa ttggtttaatatttgtctag 52560 atgtgatcta agttctgaat atcccagggc attgtgggtg ctcaattaatgtttgtggaa 52620 ttgtaactgt atttgatatt atttctattt tgataatagg catctgatgggtttaattaa 52680 agctattttg gttatgggta atagaaccca tttcaggtga tgaaaagcagaaaagagaat 52740 ctattataag ggtacagagc atctcatggt acccaaagag gggaactccagtttggcttg 52800 aggaggcagt tggaaagcta gcaggaacta aggctagcta ttctctcagtcactcagaga 52860 acccacagtt tcttatgtgc actttttccc cctctgcatc tgttttatttcttacttcct 52920 ctcagcagac tggttttctc tacttctccc tgtgcatgcc agaaaatggctaccataggc 52980 tgcatcacat cctttctcta ccggctaatt ggttaaccta gctttaacttcatagcagag 53040 taaaaccgat cggcccagtt tggatcaggt ggctagcagt ctaatgaattacttgttggc 53100 aaagcatatt gccagaacat atctctttgt taagagaaat aacttgtaattgatacttgt 53160 catgttagtg acttagtctt tgtaaagctg tgtgggcttc tttgtcgtaaaaatttggtc 53220 atctgtaaga tagtttgtcc ttattttact tattaaaata tgatttgttgggtccctcat 53280 atgtgcatag tcattgaata ggcagagcgg tctgcgcgcg tgcacacacacacacacaca 53340 cacacacaca caaaattagg cctagtgttg gattttccta ctcttaccattgtgcctgtt 53400 tttagtatgc atttgattta gcacatgata atcataattc aaagataagtctgcagcaga 53460 cttaacattt atatataaag tgtttttaag gcagaaggta ttatttaccctctttttaga 53520 agtggagaaa ctgagaatca gggagtaaaa atcagtcaac taataggttgcagatcgtag 53580 ccatagctga gttgagatat tctttacaca aattatggcc ttattcttctatatcctttt 53640 acctctatgt gagtgtatat caaactggac ttctccgaaa ttctctttggcctagtttgc 53700 aagttctctg tgtagaataa aaaattacat ttttaattga ttatttttatcaagtaatac 53760 atgcaaatga cactaaattc aaaaggtata tactttatta ttgttttatcttgagtttct 53820 tccatttcat ttcatacaga gagctaaaaa gatttaatat aaacattttctgggtcattt 53880 gtacatctgg atagcagaga actattaggt tggtgcaaaa gtaattgtggttttgcaatt 53940 acttttattt ttatttattt atttattatt tttgagtcgg agtctcactgtcacccaggc 54000 tggagtgcag tggtgtaatc tcagctcact gcaacctcca cctcccgggctcaagtgatt 54060 ctctctgcct caacctccca agtagcctcc caagccacca ccacgcccagctaatttttt 54120 ttttttgtat tttagtagct tttttgtatt tcaccatgtt gcccagagtggtcttgaact 54180 cttgagctca ggtgatccac ctgccttggc ctcccaaagt gctgggattacaggtgtgag 54240 ccactgtgcc tggcatgcaa ttacttttaa tggcaaagac cacaattagttttgcaccaa 54300 actagtataa tttcagtttc tgcaattact ttttatgttt tacaaccagcgttcatagtg 54360 agtagtatta cttcgcttgt tgcaggtttg attataaaac aacggagatggagttcacag 54420 taagtgatat gttcctacat catgaggcaa cattgatctc tccagcaaaggttttgtact 54480 agtttacata gggaaaagtg gtgggagaat tgattcgtta cactgtgctcaatattaaaa 54540 gcatgctagt ccaaagtagt ctgtgccata tttgtgtttt gagtgtgtagttgtagcaga 54600 atatcatcca tcatattgaa ttaatgttct tacttgaatt tcattgattttgttggtttg 54660 tggaatttag tttgcagatt tggttttata cttgtacagt tccgtaagaataaataattc 54720 ttagctacta tatgactaca ttttatcagg tacaagtata cattttctacattttaacat 54780 ctctgaaatc aggatgcatt tagcagtcaa ttgatggtat atcgtagtttaattggcagc 54840 ttttttcttt tttagtggta ggtgtggttt acaattagtg acttaaattagatgaaatat 54900 ggtaacttta tgtttgtatg aatttcagta acataaaaat aaaccatcatctctgtaaag 54960 gagagataat ttactttttt taagtttaag aacagctgtg taggctgagtgcggtggctc 55020 atgcttgtag tcctagcact ttgggaggcc aaggctggcg gatcatctgaggtcaggagt 55080 tcgagaccag cctggccaac atggtgaaaa ctcgtctcta taaaaatacacacacatgaa 55140 attagctggg tggggtggtg cacgcctgta gtcccagcta cttggaaggctgaggcagga 55200 gaatcacttg aacctgggag gcagaggttg cagtgagcca agattgcgccactgcactct 55260 agcctgtaaa tataactgcc aggaacttga gatttcagaa aacatttctccagagtatct 55320 ggaaaatcac ctggcttacc caaacaaaat tgccacttgt ggatatttgctaccaatgcc 55380 cctatcattc ttctctgtag tcctgtactc ataagcttag ctgcttagctgctcatacct 55440 attcttgaac tgcttctgtc actaatttct acgtgatctg attgttaagtacaaaatctt 55500 ctttaaaaag aatggtaata ataaattaat atagacattt atttatattgagtgcttatt 55560 acatgtgatt ttatgtgtaa taggccacat tggtattgta tgtatttaatatgtaattta 55620 tatttaatta taatatgtaa taaatgtata atataaaagt gaacatatgttttattagta 55680 tgataaacta cactttacaa atgtgaagct atgctcagat aagttaaataatttttccca 55740 aggatgatgg atctagtaag ttatgaacct aaagtttgat ctcagatattgattccatag 55800 ctcatgcttt ttcacctgtt tatataagac gcttctgatt ataaggtatttttgaaatat 55860 aaataatatt caacttaaga ggaatctcat ttagcagggc ttaactaatgtctaatcttt 55920 tttgtgttaa caattatctc atgaatttct tctatatgta ggtgctattctaggaccagg 55980 gcattcatag gtgaataaga cactatttct atcttaagta gctcagtctagaggacagat 56040 aggaatcatt aaaatatggt ataaaatggt cacttttgca aaatgttggtatatttggga 56100 aaaggaaagt cagtatatag tgttacatca atgagtattt agcataagttaccaaagggc 56160 ttactacagt aatattctga atctagaatg aattttgtga cttagtgtttaatacatgtt 56220 gtgtgggtcc gtttcttttg gtcactagac aagtaattga taggttttcaagtgattaaa 56280 gattgatgtt tcttgttgat gacgtctttc aaaatcatat tcttgaggatatgaaacacg 56340 attaaaaact tggttggtgg ggattattat tattatttat tattattattactttttgag 56400 atggagtctc gctctgttgc ccaggttgga gtgcagtggc gtgatctcggctcactgcaa 56460 cctccacctc ctgggtttaa gtgattctcg tgactcaacc cgtgagtagctggcattaca 56520 ggtgcctgcc accatggcca gctaattttt tttattttta ttagagacagggtttcacta 56580 tgttggtcag gctggtctca aactccttac ctcaggtgat ctgcccaccttggcctccca 56640 aagtgttggg attacaggtg tgagccaccg tgcctggctg gattattattattatttatt 56700 ttattttatt tttttgaaac agagtttcac ttttgttgcc caggctggagtgcagtggtg 56760 caaactcggc tcactgcaat ctccgcctcc cgagtccaag tgatactcctgcctcagcct 56820 cccaagtagc tgggattaca ggtgcctacc accatgcctg gctaatttttgtatttttag 56880 tagagactgg gtttcaccat gttggccagg ctggactcga actcctgactgcagatgatc 56940 cgcctgtctc agcctcccaa agtgttgtga ttacaggtgt gagccatggcatccgaccta 57000 tcattttttt agcttggtca tccaggctgg agtgcagtga tatagtcactgcggccacaa 57060 cctcctgggc tcaagcagtt ctcccacctc agcctcctta gtagctgggaccataagtgt 57120 tcaccacctc ttctggctta tttttcaaaa ttatttgtag agatgaagtctcgctatgtt 57180 gcccaggctc gtctcaaact cctgggctca agtgatcctc ctgccttggtctcccgaagt 57240 gctgggatta caggactggc ccagtaggga ttttaaggat ttttttttttaaatcaaact 57300 cttttaaatg ttctccagat gctgtcttta gtgacttgtt atactaaaaaatgttctact 57360 tattgccttc taatccatgc cagtagttat tactaacatg cccagatacattaaaccata 57420 acaatgccag tttctgtttc tgtttgtatt ctgaattttg aactgcctgaatcctccact 57480 aggctcctct aatttccaga tcatgaaagt ttatgttctg agagtgctgttactccaaag 57540 aagattcatt tgcatttgaa tatgattgtg acctcactag caagatgacaaataacctct 57600 tctcaaggca gagtagattg gctgtgttac atgagaaagc tcctttgcttttttgatact 57660 tagaacagtg ccttaagtat agttggcttt taataatgtc tctcccaatttctctcttgc 57720 ctgtttgccg aggcagaaaa ttctagttag aatatttctt tggagttacttaaatttcca 57780 ggaatatgca gatactcttc tgttaatatt tgtgactatg cagatatccctctggtttat 57840 gagatgtgga tctaaaattt acttataacc taaagtagct taggttttgtctcctaaagt 57900 agcttaaatt ttaagaagat acacagtggg gccatgtaaa aaaccaaaaataaactttaa 57960 aaaattgtaa gaagaatatt aagaaatata gataacatcc aaagattttcgtgttttggg 58020 aaggggttga gtttttttgt tttgttttgt tttttgtttt tttgagacggagtctctctc 58080 tgtcgcccgg gctggagtcc agtggcatga tctcggctca ctgcaagcaccacctcctgg 58140 gttcacacca ttctcctgcc tcagcctcct gagtggctgg gactacaggcgcctgtcatg 58200 acgcccggct aattttttgt atgtttagta gagacggggt ttcaccgtgttagccaagat 58260 ggtctcgatc tcctgacctt gtgatctgtc cgcctcggcc tcccaaaatgcttggattac 58320 aggcgtgagg caccgcgccc ggctggcgtt gagttttaaa atatgcagtaactcttacaa 58380 tccagcaaaa aaaacagaca aacaaaaaac aaaaacaaaa atgagcaattcagaaagatg 58440 ggtaaataat ataaatagat aattaataga aaaatatgca aatgcccaataaatatatat 58500 tactttacaa aataatttat tttcaaataa ttccaaattt gctgtataaatagttcatgt 58560 aatctccttc accagactgg ccagttgtta acattcgatg tcatttgctctatatctaat 58620 ttatcttttt ctctgtgtgt gtgtatacac agagagagat atatacacagatttgtatgt 58680 gtgtgtatat atatgtgtgt atatatacac atatatattg tatacatatatgtgtacata 58740 tgtatgtact cattatcatt acgttcaaac tttttaagag caagcttaaaacacaatgtt 58800 ctgtcaccca ttgtactcca gtgtgtaatt cctgataaat agaacagaataacttcaact 58860 tcggtctgtc tactttttct catttccagt tactcttggc agatataccacagaaagttg 58920 ctcttttgat actacagtgc aaaacgttgc acaattacac cactaaaaataattcagtag 58980 tattcaaaga tgatgtgaat attctgttca tcaaaccacc tgtcagcttagcatcttttg 59040 atagctctct gaatcagcca cctttatgat ggttgccaaa cgctgattctctgtcagttc 59100 tgttacagtt attaacattt tagtataagg aaaagcttta tcttttctacattttaaaac 59160 attcattcat tcattcttgt caatatggac tcacggattt catttttactgaatgcattg 59220 taatttgtta ccaacttttt aaagtgagat ataattaatt tactgtggaatacacagatc 59280 ttaagttgat cagtttgaca tacatatata tccctaagga gataatagcacaaataatat 59340 ttattttaat gctcaaactt tcccccattt gaccagtaag aggaaaaccccttcaagatg 59400 cttcctgtgt ctgtttgaaa tgtccctatc attaatcgca ccatttctttactttctgac 59460 acaagatgtt tcagattctt cttgtgcttt tctctgtcca accttgaaatcaaccatttt 59520 tctaaggatc ctaattcctt ataatggaga gagttattta gaaatcaagacgtgggccga 59580 ggcgggtgga tcacgaagtc gggagtttga gaccagcctg accaacatggtgaaatcccg 59640 tctctactaa ttagctggac atggtggcga gcgcctgtaa tcccagctacttgggaggct 59700 ggggcaggag aattgcttga acctgggagg cagaggttac agtgagccgatatcacacca 59760 ttgcactcca gcctgggcaa cagagcaaga ctccatctca attaaaaataaataaataaa 59820 tcaagatgta ggctattgct gtttgttgtt gttgttactg ggctgtcttttggttgctct 59880 tagtactttc tttttggaca gagctaggaa aatataggga cacacacgcacacctagaag 59940 tacttgttta tttttctgtg tgtgtgtgtg tgtgtatgta tatatacacatatatatata 60000 ttatacaatc atgaattcaa accaaatttc caattccagt ccaatatttagacttctctg 60060 tagtctgctc ccttccttcc attttaactt ctttggagtg aaaaacatggcccccactat 60120 attcagtgta tttacttatt tgattagtcc atttacttat ttggtccgggtaatggatct 60180 tccagccttg cagcttatct cctctgtccc tattcagctc tcatccatgtcctccgccac 60240 aggactgcac ctccatgtgg ttcccccagg cctcctcttc actgccttgtatattcagct 60300 tctgtccttg tgcctattca acccttaccc cttcacaaat ccatgtccttagtgccacat 60360 gaaaaaggag agaagaaatg gccccagaga atattttgaa gttatattgcatggttttct 60420 tttcttttct tttctttttt tttttgagac gtagtctcac gtcactcaggttggagtgca 60480 gtgactcgat ctcggctcac tgcaactccg cctcccaggt tcatgccattctcctgcctc 60540 agcctcccga gtagctgggc ctacaggtgc ccaccaccat gcccggctacttttttgtat 60600 ttttagtaga gacggggttt caccttgtta gcctacaggc acccgccaccacacccggct 60660 aattttttat atttttagta gagacggggt ttcaccgggt tagccaggatggtctccatc 60720 tcctgacctc gtgatccacc cgcctcggcc tcccagagtg ctgggattacaggcgtgagc 60780 caccacacct ggccatggtt ttcttcattt cctcgatgta ttcattttttttatattccc 60840 tcccaccaaa ttgtgcattt taagcttggt ttttaaatgg tacactgtttggtatataaa 60900 tgcaagtata ttttgttcat tcatcttttg gaattatctc cattattttaatagttcatt 60960 gggattgcta tatataaaat aggctgtttt catagattta ttgtcacgtggtttgtgttt 61020 aagtacattt attaatgatg gttttttgtt ttgttttgtt ttgttttttgacagagcctt 61080 gctctgtcaa cccggctgga gtgccgtgtt gcagtcatgg ctcactgcagcctccatttc 61140 ctgagctcta gcgatcctcc cgcctcagcc tccttagtag ttgggaccatacgtgcatgc 61200 cactatgcct ggctaatttt tgtatttctg gtagagacgg ggtttcaccctgttacccaa 61260 gctggtcttg aactcctgag ctcaaacaat ctacacacct cgggctcccaaagtgctggg 61320 attgtaggtg tgagccattg cacccagcct actgatggtt tttatgatcaaatttaatac 61380 tcttgttttc tgattcttct ggctacattg gcactgtaac caatttgaatccctcacttt 61440 ttaaaataaa ctacctcaat aaaatgtcat ctagcatgtt ttgctttttaaaaaccatct 61500 cgaataattg agtatttcct agtcagtgtc tgtgttagcc agccatttgaagatttgatt 61560 taactattgt ctttctaaaa cttaatttat gtatttgatg tgctgcagcccagagacaca 61620 ttagagatta ctgttgattc atttgtgcac ttctgtgctt ccttggcattttggcatctt 61680 gaaggattat tatccactaa tagctaattt taaaattgct tatgctttttagtgttccca 61740 tggaattcag tgttttttag gtgccactta ggacatttcg tagactggctaggaaaataa 61800 ttatttaagc aatgctggaa aactgtgagg tagctgttgc cttgacaaccagaaaactgt 61860 tctgtttgct caacaaaggg atggtaattt agtagtttaa tttcctttttggcatggagg 61920 tgctacataa aaacatctta ttttagcctc tgagatgatc catactactgatcagaatta 61980 gagaagcaga gcaaaaagaa aaggcaagag tttttttttc tggctacgtatagatagaca 62040 tgcatattta tcagggttgg tgttggtcaa aagaatctta gttaacctgctgacataatc 62100 ttttttatgt ctctaaattg gaccaaagta aattaacctc aaaatactggtgcaatcttg 62160 gtacactaag gggtcgtgat acacttttat tatggagcat ccccacaaagatttaagatt 62220 ctcttgccgc tggaattcaa catgattact tcatgtctgg attgaataagggagaaaata 62280 ttttaaagga acccctcttc cccacgtata tacacaatta cattggcatctgctctattt 62340 atgtgggaat tttttttatg acttaaccca tttgtatata gacttaaacattttttttta 62400 aaaaacaatt gcctcccagg ttgtaaagta aaaataatag cctttatttggcaatgtgtg 62460 ctgggccctc ttctaagcat tttgtgtgca ccgtcttact cctcatccttgtgacagaac 62520 tgttattgta tgctgaggca cttagctaag tggcttagcc caaggtcacctcactactaa 62580 gggatagatt tgaagctagg catctagttt aggaacatat acccttttttttcttttctt 62640 ttttttttga gacaaggtct cagtgtcgcc caggttagag tgcggtggcgcgatctctgc 62700 tcactgcaac ctcagtctct caggctgaag tggtcctccc accttagcctccctcatagc 62760 caggactaca agcaggtgcc accatgcttg gctaattttt gtatttttagtagaaacagg 62820 gttttgccat gttgcccagt ctggttttga actcctgggc tcaagcagtccgcccactta 62880 ggcttcctaa agtgctggga ttacaggtgt gagccactgt gcttggcttgggaacctata 62940 ctcttaatta ctgtattata ctgacttttt tttttttttg aaatggagtctcacagtgtc 63000 gcctgggctg gagtgcaatg gcacaatctt gactcactgc aacctctacctcccaggttc 63060 aagtgattct cctgctttag cctcccgagt agctgggatt acaggcgcccgccaccacac 63120 ctggctaatg ttttgtattt ttagtagaga gggggttttc accatgttggccaggctggt 63180 ctcgaatgcc tgacctcgtg atccacctgc ctcagcctcc caaagtgctgggattattat 63240 aggcatgagc caccgtgccc agccgaaaac tttttttttt ttttgagacgaagtttcact 63300 cttgttgccc aggctggagt gcaacggcat gatctcggct cactgcaacctccgcctcct 63360 gagatcaagc gattcttctg cctcagcctc ccgtgtagct gggattacaggcgcccgcca 63420 ccatgcctgg ctaatttttt atattttaag tagagatggg gtttcaccatgttgaccagg 63480 cttgtctcga actcctgacc ttcaggtaat ccacccgcct tggcctcccaaagtgctggg 63540 attacaggca tgagccacct tgcccagcca aaatgtggtt ttgcccctcgaatattaaga 63600 agaaataagg aagggaacca aatctgaatt actattgata aaatgacttgtttgtccaca 63660 tcaagtgttt cataatatta attataactt cctagctgac tacctcagacatcacaatgg 63720 agtgctttct aatattgact ctgttttttt gatgtggcag ctgaagcttagagaggtgag 63780 aggttcagta gcctacaaaa actcatctag ttggtgtaag gagtagagcttggattagga 63840 ccttgcctct ctgctttcaa agcctgtgct attaatcagt ctgctctattacctcatgtt 63900 aaagtaatga tagagtgata ccttatgccc agctaaaatt acatactcaaatctgaccac 63960 ctcagttatg gatttgattt gggtttctgt gtaaagtctc attcatattttggttggcat 64020 tgaaaaatag tttgacagtt tcattctaga tgattatata tgtctcaaaacttgccgttc 64080 tgaccacctt tgtaatgcca gtgctttgat ataggcttgc taaacaattatgtgtaagat 64140 tcaaattatt gtgctgaatg agaatttagg aacagaaaag taacttacctgagataatgt 64200 ataataaata gaggtggcag cagtaagatc agaacacaga tcactgctttttctgtgctc 64260 tttctaacga attaacactg ctctgatgat gtgtttcagt tttagcagcttttattagac 64320 tttggtttcc ttctgcaagt cttttttttt tgagatggag tctcgctctgtcacccaggc 64380 tggagtgcag tggcacgatc ttggctcact gcaagccccg cctcccaggttcacgccatt 64440 ctcctgcctc agcctcctga gtagctagga ctacaggtgt gcgccaccacgcccagctat 64500 ttttttgtat tttttttttt tagtagagac ggggtttcac cgtgttagccaggatggttt 64560 tgatctcctg acctcgtgat ccgcccgcct cagcctccca aagtggtgggattacaggtg 64620 tgagtcccgc gcccagccct ccttctgcaa gtctttctaa aggcattttctttatatgcc 64680 ttgtaattcc ttttcctgtg ttctagcatg aaggaaagaa aatattgcccttcaaaagaa 64740 tagtttgccc cacaataatt tgaagtaata aatacccttc tcccgtcaacacggattttt 64800 atattgttga agatgtggga tgggcttaat ttgggggcgg agggagtcccctttctcaaa 64860 ttcagcttta ataaatatgc ccaataagca aatctgcagt ttgctgtagttgaaatgttg 64920 ctagtgtctg tgaatgttaa tgaaaagaat acgcaaatgg gtttctgaatactaatagtc 64980 taaagatgtt agtattctat accctatttt tgttaagaat tatctattaaaaatttaaaa 65040 gtgtatcaat tcctaaattt ttatatgttc ctcagtaggt acaaatatgcaacttttagt 65100 tgatttattg ttctcttcta tgtttcataa ttttagttgc ctcatcagttttaatttatt 65160 tttaactata ctgtttgtct ctgaaaataa aattttactg gctagtatggcagaatgtgt 65220 taatagagga ggttgcaaat tgtggaaagt tatgagcctg tggtcatgaagactgccctg 65280 ccatttgggt ttttatagca gtaataccca gtaccaattg aaccacaaagtgaactgaaa 65340 tttccttaaa ttgtttctct ctctataaaa catttaaaaa aaatttaattgatgagtaaa 65400 gcttgaatat attcagtatg tgcaacatga tgaattgatg tacattttatgattaccaca 65460 gttaaattaa caaacacatt cattatcact tgtgctgtac attatattcccatccttatg 65520 ataaaaattt ttttttaatt aattgagata ggatcttgct gtgttgcttgggctgatctt 65580 gaactcctga gctcaagcag tcctgcttcg gcctcccaaa ttgctgggattacaggtgtg 65640 agccacaatg cgtggccatt tttatgattt tggagcgaag tggtgtgagtcccttttcat 65700 attattgctt aaataaagcc actagtatgc ctggaatgta accataattttggccaggga 65760 aaatgatatt ttaagagacc taaggcaggt aggtaaagta gaaatgcatttattcactag 65820 gtacaacagc aatgtaaagt tatcaagttg tagttaataa tataaaaaaattagaaagta 65880 ttagtgcaga aatgattatc tttctgtaag ggtatattcc cattatggtataaatgaact 65940 gatgaatagg cattttccta agttgatttt aaaaattgta taactgatatgttttgagtt 66000 tactttttat gaaatacatg gaatgtgaag caggtggcca ttatggaggtctgatataat 66060 tgtgggcact cctgttatag cactagaata ttgttttttt ccctttttctttgggaaata 66120 gattactctg tatgatagct acaactttta ggggagaatt tattttaaaatctaaatgaa 66180 attattcttt cattatttat tactcgtaac agcatttctc actttatattctatgggatt 66240 tctgtaggat gttaatagat gtcaatagaa aagaaagggt tctttggacaaatagattgg 66300 gaaatgttga gttaaaatgt aatcattttc tctattatag ggtttctcagatcttttagc 66360 ttacatttat gaatctccaa agttgaggca gagaagtaat tcattcaatatagtgaattt 66420 cccccaactc ctgcctcctt tccttttggt ggaacgtcct gggatgttagtactttttat 66480 aaagcacttt ggaaaaagct tacttccatg attttccttt ggctatggaattactataag 66540 cagaagaact ccttagaaat aaaaatgtag aaatgtacct gaatgtaaaacgtaagagca 66600 gctattaaaa actaaatcac aactgaggaa acataaatat tagttaaggtagaagagtaa 66660 atatccagcg aatccctcct attaaagcat ttaagatatt ttggcttcaaagttttgaga 66720 ctttccagat gactttcttc atatagtttt cactagtagt ttaaatatactaacttttct 66780 cccttgtatg ctatactaca ttcttttttg tttgcttttt tgagacagggtcttactctg 66840 tggcccaggc tagagttcag tggcacgatc acttttcact cgagtctcgacctcttgggc 66900 tcaagcactc ctcccacttc agtctcccaa gtagctagga gtacaggcgcatggcaccat 66960 agccagccag tttttatttt tttattttgt agagttgagt tttccctatgttgcttgggg 67020 tggtctcaaa ctcctgggct caagtgatcc tccagccttg gccttccaaagttctgggat 67080 tataggcagg aaccaccaca ccaggcacta cactaaacta ccttagacttttctagatag 67140 agatataaca gagctattct gaatctgggt gactgccaag atacagcctaaatattagaa 67200 gatttccccc ccaccccaga atttgagctt taaagatctg ccttctggccaacggaccct 67260 tttcttgtaa gtggtcatag aataaataaa tatttgaatg aatgagtgactagaggggta 67320 tacttgtgag tgagctaatt catatcatgg accatatgta tttaatggaaataagatacc 67380 tttatatttt atgtgatgta catttaaata gtctttatga taaacattttacttgttttt 67440 taatttatag tattttatag attggtcttg ttaacataca tttcatatttataactgttg 67500 tagcttaaca agattaggct cactattctg aggtctgatt atgggaaagaattagagagt 67560 ttaaatctaa ttatatttct ttgcattttt aggcccttga tgagcctccctatttgacag 67620 tgggcactga tgtgagtgct aaatacagag gagccttttg tgaagccaagatcaagacag 67680 caaaaagact tgtcaaagtc aaggtacagt atttatagat ttcataaattgtatgttcag 67740 catttgatat gtaaactttt atttggtgag tgtatttaag atttttctgcaaaataagtt 67800 tctaggagtg aaggttgtaa aaatacctca aaacaaagca ttttttgttattttaaaagt 67860 attataattg gaaagaaatt agattatcac agttggtatt ttgaatgccatttcactata 67920 gacgtcttat tgtttgtata gtgttttaga gctggaaagg aagttagaagtcatctagtt 67980 taattgcttc attttattac aagaaaaatg aggctcggag agggttagtgatttctccaa 68040 ggccattatg ccaaattcat ggcagaattg gaactaccat agttcatcaattttaaggca 68100 tcaccccctc tatttcagcc ataaaattag aatgtgactt gtaattgatggtgccttaga 68160 taacagtgaa ataaggtaca tctagttgtt gactccttat gctctgcctttcttgaaaca 68220 tctttctaaa gtacttgagt tttgtcttaa tatagaaaaa ggagtgtaattgcttccatt 68280 ttaataaatt atcagcactg aaaaatacca gacaagtgaa cttgaatatagacatttatt 68340 catttgtcaa taatttattg agtgacaaac cttttgcagg gaacttttaggtgggaagga 68400 ggatggggat attaggatga gattgacctg atctgtcctc aaggaatttataatctagat 68460 tagcagatac cttttaagcc attggttttt atttcaccaa ttacatagactgtagaggtg 68520 ttgtgcttct gtggagggta tcagtctctt ctggagcttt tactgttcctatattcctta 68580 tgcatggatg tgttgaaaaa gctacctggg tgattctaat ttacactcctggttaggaac 68640 cattgtgata agtcctttaa attaactaca aactggtagg gggctagtcttggccccacc 68700 ctaccttcag tttccacatg cttggtggtt ggtgctttta tagtgtgcatttcctgttct 68760 ggcttcacac actggtattc cccaagcact gttttttgcc agtactatcctgtagaggaa 68820 gagaaagcct tttatcaatg tttgtacttt ttcaggacta ctggtagcacatgtatcacc 68880 tttgtgtgaa gtagacaaaa gccctctctc ctggtggaaa gccttcttagcagcgcttgg 68940 catgcagata gcaagtttgt gccagttgga aaagacattc tctttggactggagctgtgt 69000 cactccagga cccatgcttt ggcccacaaa gcaaaggctg gatttaagccccatcttgca 69060 tccttagctg cacatttcct gtgtaatgaa tgaactagga aaatgacatgtagcagtcct 69120 gccatttctt gctgtaaatg aaaggaacaa aactcttact ggactctactgcctctattg 69180 atagcacaga gtaccttgtg ctttataagt aatgaatatc taaagcttatttttaatttt 69240 ttcggctgcc ctaaaaattt gtttctgtgg aggtgatgtc cattaaaacacatacacaag 69300 gaaaaagaaa gtgtatttca ttcagtgatt ttgtcagtag atatagggatatatttttta 69360 acatgtgtaa tttctttatg attcaaagtt aatatatttt taacaaatttgttgatatgt 69420 tttcactacc tgttttcatt tgttgaacat ttactatgtg ctaagctctatctaaaggtg 69480 aaactctagt gttaagacct agtctccatc tgtaagtagc ttatagtttaagtgaagggc 69540 acagatatct aaacatgtga tttaaataaa aagtgtgata atgcttttgtatgacacaga 69600 aatgcctggg atgctgtggg tataaggcat ctcctttcat agctggtttttgagtgggcc 69660 tgcctgttac ctaaaataat tatcacatat acctgataaa gaccatgttaatgattcaat 69720 ccctataaga actcttttcc cttgttagag aaaacaattg ttttctatactgagtagtgg 69780 aagctatctt gtttttcacc agctaattaa gtatttaaat ttgaactcattattttaact 69840 tactcttttc acctctggtt tcccattctc tgatttctgt aatgatatagagcacagtaa 69900 agttctggaa ctagactgcc tacatttaaa tatcagctcc accatgtctagctctgtaaa 69960 cttggaccag gtacttacac tttctgtttt ttgttttgtt ttgttttgcttttgagacgg 70020 agcttgccct gtcccccagg ctggagtgca gtgacgcaat ctcggctcactgcaacctcc 70080 acctccctgg ttcaagtgat tctcctgcct aagcctccgg agtagctgggattaacaggc 70140 atgcgccacc atgcctgggt aatttttgta tttttaatag agatggggcttcaccatgtt 70200 aaccaggctg gtcccatctc ctgatctcag gtgatccacc tgcttcggccttccaaagtg 70260 ctgggattac agggatgagc caccgcacct ggcctgtttc tcagttttcttatcaacaaa 70320 atgggaacag tcatacctac ctaatagggt tgttgtgaag attaaatgtttggtactaga 70380 gactgatgct cagatctcag ttaagtgttt gctgctactg tttattattaaggtaggcct 70440 tcacaaaatg atataatacc taatagccta aaatccccat atctttctttctcatttttt 70500 aggaaaagca attttacaac taaattcata actgatagta ttgaaataaaggatgatatg 70560 tgggaccagg cgtggtgact cacacctgta atcccagcac tctggtaggtcaaggcgggc 70620 ggatcacttg aggttgggag tttgagacca gcctggccaa tgtgttgaaaccccatctct 70680 attagaaata caaaaattag ttggcctggt ggtaggtgcc tgtaatcctaactacttgaa 70740 agactgaggc aggagaatca cttgaaccct gaggtggagg ttgcagtgagccaagattgc 70800 gctactgcac tccaacctgg gcgacagagt gagactgtgt atcaaaaaagaaaaaagtta 70860 aaaaaaaaaa aaatgacatg tggaacatct agtaatttag atgtgctatcattgattact 70920 tttatttttg aaaacagata cggtctgagc agttgtctgt aaataattttttagttaatc 70980 tacttaggga ttgggatgtt gtataaacta ggcagctatt attttatatttcttttacta 71040 caaatgaata attggtgcat tgatgagctt gtgttgcttg gtttatttttggtggctaat 71100 taaacttata atctctaggt gacatttaga catgattctt caacagtggaagttcaggat 71160 gaccacataa agggcccact aaaggtaatt catgtattca ttgttaattctaatggttgt 71220 ttgggaaaaa ataatcatac ttggtattaa ttcattggct cttttgttattgagttgata 71280 aattcaatat gtaattttct ctaaaaagac taatagaaaa aatagacttaatctcagtga 71340 ctaaggaggc tgaggcagga ggatagcttg aggccaggag ttcaaggttgcaataagtta 71400 tgattgtgta cactgcactt ggggacagca tgagatgctg tcttttaaaaaaaagattta 71460 atattaaata atacttgata tggtagtaaa accagtttat tcagaaataaaatgagtatc 71520 aaagaagcat tgactacatt tatttactta atccaccata tttccattactaaaatgatg 71580 gattttccaa aattaataga tgatgaacta gggattagtg atgtcagttccacgatgatc 71640 tgaaaagtga gaaagttaga aaaccaaaaa aatcggagct cttacttctttggagccact 71700 actggcaaat gaaatctttt aagggtcatc agcctttaca ctaacagttggagagtttta 71760 tttctttgca ttcatttttc ctcattggca agtgaaataa atgctgctttgctttttgcc 71820 agtgagcaag ctaaaagaga atgtgtgggt aaaccactat gtgctctttggctggtttag 71880 ggccaaaagg aaaccccaga gcaattttta aattgtccct atccttcctgaggttccttg 71940 aaagaagagt ggaaatacgg ggcaaggaca gtcagtttca cagatggtggtactcacggt 72000 gtagtctact gaccctgggg attcccagga ttcttttggc gggggttgtaagggtaaaac 72060 tttataatag tacttaggta ttatttgcct tttttactac attaacattttcactgatgg 72120 tacaaaaata ttgatcggta aaactgctgg cacttttttt tttttttttttttttgaggc 72180 agagtcttgc tctgtcaccc aggctggagt gcagtggcgc gatctcggctcactgcaacc 72240 tccacctccc aggctcaagc aattctcctg gctcagcctc ccgagtagctgggattacag 72300 gcgcctgcca ccacgcctgc ccaatttttg tacttttggt agagacggggtttcaccatg 72360 ttggccaggc tggtctcgaa ctcctgacct caagtgatcc acctgcctcagcccctcaaa 72420 gtgctgggat tgcaggcgtg agccactgtg ctcacgccaa ctgctggtacttttaaatga 72480 agtcaatagt cattttattc ttcaccatca tgcactcagt taaaagaaaacaaacccagg 72540 ttttggtttg ttaatttacc ttttaaaagt ttttctaaca tgtttaaaaatactttaggt 72600 aggagctatt gtggaagtga agaatcttga tggtgcatat caggaagctgttatcaataa 72660 actaacagat gcgagttggt acactgtagg taagaaaata aattttcttttaaaaatgtg 72720 tttttagtta cacaaagaaa acttaataca aataaaatga aatttcctggattcacttct 72780 taatacccta gatrttatta ctgttactgg attttatctt tccacatcttttaaaaattt 72840 atttccaaag tatatgtatg tatatgtaac aaaaattgaa attatttaatgtgtatatta 72900 tgcaattgga attctagctt tttttacttc aaagtatatc ttaggtaaatttcttttttt 72960 ccccaatctt acctattttt aagtttatag ttcagtaatg ttaaggatattcatgttgtt 73020 ttataataga aatgtctttt aactgtgtat atatatatat atatagagagagagagagag 73080 atatatctat ctatctatct atctatctat ctatctatct atctatctatatagctccca 73140 gcaggaaaca aagtagtagt gtattttata atagttgatg tgctagtttattttttctta 73200 ctgatgaggt ttccagtttt cttactatta caataaatgc tggattgaatgtctttttta 73260 cctaattagg tacttaatta ggatacctac tgtatcctag tatttctttagtgtacatac 73320 tgagaaatag aggttgaaga aaaatatgct ccaaaaactt tagtagttaatgaaaactgc 73380 cttcctaaaa gacactgtct gttttctcaa tccttgtcag attttatgtcatttaaaaaa 73440 attttttgct aatttggaca taaagtagta tctcagtttt gtttcagtatgcccttccca 73500 tgattactag tgagattgaa catcttttca tatgtttatt gaaaggaacataatttcatt 73560 tgtgttttct ccgttttgag ttgctggctc ttttattctt tccctttttctcttgagatg 73620 atttatcttt ttcttattag ttttggtata tgtgttcttt atatattttggatattaact 73680 gtttactact tcagttataa atatatttct cctgttgctt tattcttgtctctgctttta 73740 attttcatgt agtcagtttt tgtatggtca ccttttattt tgtaattttagttttgaatc 73800 ttaaagcctt ccatcagtgt tataaatcta ttttctaatg tattcttcaaattttttata 73860 ctttttaaat ttatttttat ttttgtttta caaacagatt tttatttcacctacagtagt 73920 atgtattttt tccaacattc taaaataatg attaaaaagt atttgtcctagattggtagc 73980 catttattcc agtattattt attaaaggta acaattctta ccatttaatgacctcagaga 74040 tgaaagtaaa cacctagtaa aatgtctttc gtgatttcag agtttgatagaactttcaga 74100 tagaataggt gagttttgtt tatacttctt tggtgtgaga ataaaacctggtgaaaatag 74160 agggaaaatt atttgatttt gagacataag gaaatatttg actttatgtaagtgagaaga 74220 ctgtgagcaa ttctatgtag gaagtctaga tggtgattgg ttgtaatagtctactaaatg 74280 tgacacttat gttgtatatt ttagttacat catttaaaga tgtactgaaaattatttatc 74340 cagtttcaca taatgtaact tgttatgtaa cgtataagaa tctaattttagttcagccag 74400 taaggaaagg gtattggctt tcttttaacc attaatcatt tctcaataaacgtgagatcc 74460 tgttgagcat cagaaaaaga aaaggaaaga agagtatcta attttagtaggtaggcagaa 74520 aatgtaattt ctaaaataga gatctggtaa cattatttaa aacagagttactgtcttccc 74580 atgatttcta aggaatgtag catatcccta aattatttat gtatatcacagatttatgca 74640 tataatacaa atagtcatac attccatatc tgttaactta tggtggttttgctggggtgc 74700 tttaagtact ttatttttat taaaattttt cccctacttt attgagatataattgacaaa 74760 atcatataaa tgtatatgta acatgatgat ttgataggca aatatattgtgaagtaatta 74820 ctgcagccaa gttgcttaac acttccatca ccttttaata gacattttagtattttaagt 74880 cgctagtttt caactgggat gaatttaccc ccagggaaca tttggcaatgcctggagctg 74940 gagacatttt aggttgtcac agctgggaag ggggtgctac tgacatctagtgggtagagg 75000 ccaggagtgc tgctaaatat cctgcagtgc acaggacaag cctgccacaacagagaattt 75060 tctggtgcag tatgtcacta gtgacagtgc taagggtgag aaaccctactttaaataatg 75120 aaaatacagt tattttttta aaggggcagt ctgattgcat atacttgtaatctttcttag 75180 gtaaatctgt caatatctgt cagtaggaaa acttggcatt tctttaccattttgagaaga 75240 gttaattagc atttaaatgt tttcctcttg aaataccaac ttcttatttttatttggtat 75300 atcctgtttt atacaagatg ttttcatttc atatatgttg tttctttttcactagaaatt 75360 ggaaaagtta ctttaataac tctatatttg aagttactca ctagaaaacataaaatgtga 75420 attgaattcc aaatgtagtc ttaaatagta gatctgtttg cactcagaaaattgtaatac 75480 atgtcatctc tttgatcttt gaaaaaatcc ttcagtagct tccatttttaccccctctca 75540 tagtttttga tgacggagat gagaagacac tgagacgatc ttcactgtgcctgaaaggag 75600 agaggcattt tgctgaaagt gaagtaagtc atcatttaac aaatgaacatgtcttaatat 75660 tttttattgg gaggaataaa tttttgcttt ctcaactcag taaacctacttcctaatcag 75720 gaaagttcat taatacacaa tacccttatg atgtaatctg tgaagcctgtttaaggccct 75780 gtgtttgagc accaggttct gtcttctttt ttgtctatgt aaagtgtttcagtatttgtc 75840 cacagagaaa atacaatggg ctttaattat tatctttagg cagtttttaaacagacttaa 75900 aatttgtaaa taactagaaa acagatttct gatttgtaat ctacttgatccttgtcgtag 75960 ttaaaggatg tgtagataca acagtcttta actactgcta ctgctattagctgatattat 76020 tatgcccttt cctcttgcct gactgccctt cctaaaattg tcccttccattccttatcat 76080 cctttcagat tgatttaggt cttattccct aacatcaaaa tatgagtacataattattta 76140 ttgtctgcct tcctcactag aatgtgtcac acggtcagag actttgtatatcttcttcat 76200 ggctttgttc ctagttgctg taacagtgcc tgatgcagag tatgaacttcataagtgtta 76260 gttggatgaa tagatgaggt gcttataatt gccagggatt ttgctaagtgctttacatac 76320 attattgtat ttgcaacatt atgaggtagt tactattatt atccccaactttcagattag 76380 gaaatggata gctagagctg gttttctaac cagcactttc tagcaccaaagccatatcct 76440 taaattgaaa gatgttacta agagcagagg gttcacaaag ttttgtaactaactcgcttg 76500 caattcagaa tttaaaaggt agatgttgct ctaaatggtg ttacgttctctaatccagtc 76560 tacagatatt cattgataca cagtgtggcc gagctatttc ttttctctactcactatttg 76620 gaatagtgtt tctttgggga tattttaata ttactaatta atattacttatttgttatta 76680 tttattactt taatattact ttaatattac ttatttagta gtgttattacttaaacacta 76740 tttggaatag tgtttctttg gggatatttt aatattactt attaacctattagactttat 76800 gtttttgttt tgaaaaattt ttgcttctct taatgattga attcccacttaaatttagaa 76860 tttggacagg ttctctgtat tataattcca gcctatccct ttagtctcatattcaaccac 76920 ttccctgtac ttctgcctta ttagactgct tggtattttc agactatggtccatactttc 76980 ctttattgac tcctttcctc ttgttggaat ctcacttatg ttgaaggccattataaatgc 77040 ttcctcctct atgtctttcg tgagctctct acccaaattg gtcgttttgcacttctcttg 77100 cttattcttg ttttaggacc tttttttttt tttaactttt ctcatttttgctgtaagatt 77160 ttaagctgtt ttaggggaat atcctttctt tcctcacttg ataggccccccttttcctag 77220 caatttctac acaaaagcct tcaataaata tgtgtggaat aaactgaaggtttacataca 77280 ataaatacag acagctttgg ggcaagtata gtgataaagt acagaattctccctgagttc 77340 gtggaattat tttaatttca tttccgtaca gtcagtgagt atgaggacttgaagacatgg 77400 tttttctcct gttaagtgaa ggtctgcatt ttattttttt agtctagagaaatagccaaa 77460 atattatttt ttttaaccag gagaattttc agttggaatg gttcataagacagttgtaag 77520 ttaaatgctg aaggtaatat gaacaagtgg ctatgtatcc ttcatatcttcaaggaggtt 77580 cagttttagt gtgtctgtgc aggtgtgtac ttctaattta tgttttgctagaaggaaatt 77640 agaatactgg tgtgtgattc ttcaattccc ttcccatgtg cccggagctgtgtagtgcta 77700 ggtaccggga caaaggttaa gccaacccat gtaaacatgg aacttatctttgggtactta 77760 ctccctgttg gaggagaaaa agcattaatc aaaccaagcc atgtaaacatggaacttatc 77820 cttggatact tactctccgt tggaggagaa aaagcattaa tcaaacaatcaaacatattt 77880 taaaatttga gtatattaag ccctgtgaag atgtggtcca gagtgcacccaataggggaa 77940 tttaatctac catctgtatg ttcttttctc tttatttggt atttttcatattgttttaaa 78000 agttttaacg tttttgaaag accattcaga gtattagtag tagtagtagtccataacatt 78060 gtcctttaag aggaaagctg cttcatactg ccagtatttt gggtcactaatcccatttaa 78120 taaactactg aaattaatcc catctcataa acactgaaat tcaggattttcatggaaaga 78180 gatcacattt gcacttcagt cagtttatct ttttatttta ttttattattattattatta 78240 ttttttgaga cggagtctcg ctctgttgcc caggctggag tgcagtggtgcgatctcggc 78300 tcactgcaag ctccgcctcc caggttcacg ccattctgct gcctcagcctcccgagtagc 78360 tggaactaca ggcacctgcc cccacgcctg gctaattttt ttttttttttttttgagatg 78420 gagtctctct ctgtcgccag gctggactgc agtggcacga tctcggctcactgcaacctc 78480 tgcctcccgt gttcaagtga ttctcctgcc tcagcctcct gagtagctgggactacagtc 78540 gtgtgccacc acgcccagct aatttttgta tttttagtag agacggggtttcaccatgtt 78600 ggccaggctg acctcgatct cttgacctcg tgatacgccc gcctcagcctcccaaagtgc 78660 tgggattaca ggtgtgagcc accgtgcccg gccacgccca gctaatttttaaatatttgt 78720 tagtagagac agggttttac tctgttagcc agggtggtct cgatctcctgacctcatgat 78780 ctgcctgcct tggcctccca aagtgctgag attacaggct tgagccaccacgcccagcct 78840 cagtcagttt aaattccaac actgaagcca ctggttttag ccataaccagcttaatgagt 78900 atgttacctg ctgcctaaca tctgtaggac agtttaggta tttctaaagaccattcatac 78960 tttgagtatt atacaaaagt agattcatca tctgatattt atttatttatttattttttt 79020 gagacagagt cttgctctgt cgcccagact ggagtacagc ggcatgatcttggcttactg 79080 taacctctgc ctcccgggtt caagcgattc tcgtgcctca gcctcttgagtatctgggac 79140 tacaggcgtg cagcaccacg cctgactaat ttttgtattt ttagtagagatggggtttcg 79200 ccatgttggc caggctggtc ttgaactcct agcctcaagt gatccgcccaccttggcctc 79260 ccaaagtgct gggattacag gcgtgagcca ccgcgcccag ccaattatctggtattcttt 79320 ctttgtattt actatgtgca attgaaaact ctagagaagt ttaggtaaaatcccagttcc 79380 atattttctg cagcccattt taggtaacag ggccatagac attttccctctaggcctacg 79440 gagttttcta tgtgccttta atacagcttt caggagcctc tggtttttgctctttaggtc 79500 taagatggga caaattctac cctaccccca ggtcttttta gtcccttgaaacaaacatgc 79560 ccctactttc ctggacattg agatagggac agagatattg cttatgtttcttcttgttat 79620 gtactaggga gagactgtgg tctcttattt gaaaattact atgtgtgattcttaaacaac 79680 tctttagaaa ttttctttcc tgtagcaagt gaaatgacta acagatacttaaaatagaaa 79740 tcacactgtg tgatataaat ctctctctct cttttatttt tctgctttaaagagagcaac 79800 cttaattttc ggtggggaga gcatacattt aatccatctg tttgcttgacagaaagtgaa 79860 tccggattaa tttattttcc ctgtttacca aataaagtca tttagtaatactctatagtg 79920 atacagtatt accaaaataa tatttcagat tacatagaaa ttttcaccataggagttaat 79980 gacaggcaac gtctagattt tccgggtggg tgtggtggct catgcctgtaatcccagcag 80040 tttgggaggc cgagttgggc agatggcttg agcccaggag ttcgagaccagcctggacaa 80100 catggcaaaa ccccatctct acaaaaaaca aaaattagct gggtgtgatagtgcacacct 80160 gtggtcctag ctacttggga ggctgaggtt ggaggatcac ttgagcccaggaggttgagg 80220 ctgcagtgag caatgatcat gccgccactg cactccagcc tgaatgatagggtgacattc 80280 tgtctcaata aaaatagttt ctgtttggta tatactgagt ttaggacagtttttgcctcc 80340 aaataatttt tatttttatt ttttgagatg gaatttcgct cttgttgcacaggctggagt 80400 gcaatggtgt aatcttggct caccgcaacc tctgcctgcc gggttcaagcgattctcctg 80460 cctcagcctc ccgagtagct gggattacag gcacccacca ccatgccaggctaattttgt 80520 atttttagta gagacggggt ttctccatgt tggtcaggct ggtctccaactcccgatctc 80580 aggtattcca cctgcctcgg cctcccaaag tgctgggatt ataggcgtgagtcactgcac 80640 ccggcccaaa taatattttt taaatgcctg atttaaggtt cattaggacagggttaggtt 80700 ctgtacaaac taaatttgaa agattagatt ttttgcccta tgagagttgtgaaagctatg 80760 atttttgtca gtcttttgaa ggaatcaatc tttagttaat cctttatggctagtaccttt 80820 tttcttgatc atactgttcg ttgttagata catacatcta tgtaccttaaacacatttac 80880 atatattcta atgaattatg taaaaaagtg ttaatatggt tcatcttttcacagttttgt 80940 tcattgtagt tataacaaaa ttgtctcaat tttaaaaaag ttatataacatttcttttta 81000 catattacat tagtttagat gcctgctctt taattctaag ttcatggatactctttctta 81060 taccaccctg gtttgtatct ttaagaagtc tctgtcttaa atagttcctcacttttattt 81120 tcatctgggc agttttcttg gaaatctcta gttgctctga tgtttgtgaaattatttata 81180 acaggaaaga ttcttcttag gatgaggaaa ggaacagaaa ctgtggaaaatcttaagaaa 81240 acgtaggaaa ggaaaaaaat caaatgtaga aattaaatgt aatttaaaaaattaaattaa 81300 tgttacattt tattaaaaaa ggtttattgc agaaaatgtt tgcagagtagtaatataact 81360 tactccgagg ctttttgttc gtgcgattga tgctttatct tcataattaattttgtttct 81420 tattaataat tttattttat ggattatgat tccccatgta caaattaatattggagctgc 81480 agttggccct aaccttacaa atagatttct taattctcac atttgtctttccactgagct 81540 gtgtttgttt tgtattttaa tcattcttag atgcctatca accttcacataggtaatgaa 81600 tcatctagag agagcagcca aatttctttt cacttttttt ttccagtaatgagctcattc 81660 atctttattc taatatcttt gaaactagaa ttttctatct ttaggaaagctgtgttttag 81720 cttccctggg gggagagttt aatctcaaaa tctgagcata ttcttaaattgatttggttt 81780 aaaacaaaga aggcacttgc aaagtgttaa ttttttgatg ggtttatgttcttacccaca 81840 gacattagac cagctcccac tcaccaaccc tgagcatttt ggcactccagtcataggaaa 81900 gaaaacaaat agaggaagaa gatctaatca tatgtaagtc cattttcatgactgttagtt 81960 gaacctgaat tcatttctcc ttctagtatt gtttatagtt attttaaaattgtaatttgc 82020 aaaatgctaa ccagatctaa tagtaatttc ggatctaata gtaatttcagactcctcagc 82080 atgggtgatt gaggttctcc atgctcagcc aaccttactt aacagctgtatctgccttca 82140 atttctccct gcatgttatc ttgatgctgt agtcaaaatg aaaccagtcaataattcctg 82200 aatacatcgc ttgttttcta atatcatgct tttattcatg ctattatttttggtctccat 82260 gcctcccttc ctggtaactg tcttgtccat gccttaattc tcaacaaaaatgcttaaacc 82320 tttatgtgaa ccattttgtg tctccccaca ctttccttga aaccagagatgtttctctcc 82380 cctgtaaacc caattctctg gcatagtatg aactgtggag ccagttggtctgggtttgaa 82440 tcctggctct gccacttcct tgctgtgtga tcttgggatg gttacttaatcttttcatac 82500 ctcacttcct atctataaaa tttggataat aatattacct atctcttagtgttgttgtga 82560 aaattaagtg aattcatatc ctttaagtgt ttatagagta aatgttacataagtactaga 82620 taatcaacat catatactga catgaaaaat ttcatgatga aataccagtatgtaacagtt 82680 tcatttatat atgaaaatgt gtttacatgt atatataaaa attctgcttcctggtgttta 82740 tgtctgacaa ttaggactgg ttcttgctga attattcgaa ctttaaaaaatttctacgtt 82800 tttatatata ttataggaaa aatagtctct tatttgtcaa taagtttttttaatttttag 82860 aagattgcat ctagtattta gacatatttt tacgatttta tacacatcatcctaaagtgt 82920 aaaaataaat ttaaaaatga atttattgtg caatatcacg taatattgacctgcaaatgt 82980 gactaaaaat tatgatctgt taaaaagtca gttatttttt atttcctcttctgtcgttcc 83040 ccaactgtta gatgtttctt agttgaggcg tccgttttgt gaatatgagaataaagcacg 83100 catttgttgc tgaataagtt gatctttgct ttctaaagac tccagcaaacattctaagtt 83160 agtggttttc ctgttgagaa tctctatgtg taatgagaga tttgctgttgagattgtgtt 83220 atacatgtgt aaagtatgaa tgcagacaaa aggttgaaga ttactagtgtaggaatgaag 83280 acctgctcca tctgtaatct tgttttgctt gctcttcctt gcccttctctaccacggttg 83340 gtcattctca ctgccaagga acctgacaat gttactcttg cttggcgttttaagccttgg 83400 tgtgtaagca ttacaggcaa gtactacagt ttgaaatgcc catctccttgattacatttc 83460 ctcaaagttc tttctcccct ttcttcaaag tgattttctg caacaaagtcttaaacctag 83520 atcttaaggg cattcatctg accttcattt cctcttccat tactccattgaaaaaagtct 83580 ttgctgtgat ctcagattgc catttaaaat gtatttttgt acaaagatatgtaaagttat 83640 ctcttcttag gattcccagg acttcgttgt tggagtgatg tatcaccaaatttgttttct 83700 tttagcagct gttattccac gtatcagtta atctacccct agttattccaaaattgatta 83760 cttttaacat cttccattaa attgagtttc agtatattct acctttaatcctagtaagtt 83820 caagtatttt ctttttttaa tgattatcat cctctagcta aattttttttaaagcaagtt 83880 attgattctt ttaatcaatg tattactgga ttgaattttc tccttataacttacccattg 83940 tttgcagcga ttgtgtttaa gtcatccaga agaatagaat acctgaaattatggaaatac 84000 ttgtttgtcc ctctcctctt gaagttgttt tcccataatc tggcaaaagtttatgtgcaa 84060 ctgctacaat aggaaagcag tctatttgct gttatatttt tccccttcagaaaaaagata 84120 aagcataagg tttacaatga atcttgttaa caagggatta tttatgagtatggattttac 84180 ccagtaacag tgctaagatt tacccaatac ctggtttata cttgctatttaaatacaggc 84240 ctgcagatct gaacacattt ttaaattagt gtataattca tgtatcataaaatttactca 84300 tatattttaa tctgtagatt tgtatccaat acttcaccct tttaaagtgtacagtttagt 84360 ggtttttagt atattcataa aattgtgtaa ctatcatcac taattccagaagatcttttt 84420 attttgccgc tttattttaa tatgagaatt tgattcattt cctccttgataaaaattgag 84480 aaaaaaatta aaaccctttt atagttaagc gtacataggt tctgtgtgtttctaagaagg 84540 tatgtatacg ttgtcagtct tccagatact aatagaacat ttcaatttacaaagtttgaa 84600 actgagtaaa ctgatttgcc taaaataaaa aataatcaga attgcatgtcaaaggttgag 84660 cctcttgttt ctagctcaat ttgtatcatt atttggtatt ccttggcccatatcttgtct 84720 aaagttcgtc tttcttgatt tacaactaat tagagttaat agtgaagtgtgatcagcttt 84780 aagtgattgg ctgttagagt attatggtaa aaatttccat gtaattagctttctttctag 84840 tgacatcatc agattttatt tcatagagac agcgcattat tttgttgtggctctacaaat 84900 accttgccac caaagaaata acaatgtcag aaatgaataa aaatcaagcaggctctctgc 84960 ttgctctgtc tttgctcctg taaaatattt tggatgctca ttaattttagttcattagtt 85020 tattgagtac ctgtgtaaac agttttctgg ctaagctcct ctaaaaggaaaagttaagga 85080 gcttcatgtg taatagtatg gtttacactg atgtatacat tttgtgattttagaataaaa 85140 atcagaaata ccataataaa aaaataaatg catctttttt attttatgaacaagtattta 85200 tttagcatct atactgtgcc aagcattgtt caggtacttg agatatatcaataaacaaaa 85260 aaaaattact taaaaaatct tcaacttgag agcttacatt ctctcaggggaggagatagc 85320 gaattaaaca ttggattcac atacttggtg tgaaaaaaaa gaataaacataagaaataag 85380 taaattatat agtatgataa aagtgaatgc tgtggaaatg aagagcaaaatgaaaagagg 85440 tagatgggca gggactgtaa atttgaataa ggtggaacaa agtcttgagtaggtgagggg 85500 gaaggggcat ctagtctaaa aatggagatg attttagaca ggaaccttgcaattttatat 85560 gtaaggtgga tagatgtttc agctagctga acaaaggtgt tttgtttttctttttgatat 85620 actgtgaaat ctgaagtctt aacacttgtt aagtcttaag aaaagttacaatttttacaa 85680 tgatgcagat aattttgtta gtcaaataaa gcccaatttt ctctatgttgtacagatttt 85740 gatatgtgtt atctctgaca ttgtagcttt ctagctgcta ctgataatagagtgttcata 85800 aacccctagc tgaatgagtc ttctgtggct actttgagaa agcagtaattatgattttaa 85860 gcttggttgt atcctgtagt cattagacag taaaaaaagt cactactcttaagctcttgt 85920 aaatcaagta gaaatcagac ttgtgaggac tgtcgttact gtcccaagctgtgtgtctat 85980 gtgtagggtc ttattctcca ctaatgtaaa ggtggttgag ttttcttattgttacctact 86040 ttctaagtag agattgaact tctttaagtg ccattgaagt acaatacatctttttttttt 86100 tttttgagac ggagtctcac tctgtcgccc aggctggagt gcagtggtgcgatatcggct 86160 cactgcaacc tccgcctccc gggttcaggc aattctcctg cctcagcctccagagtagct 86220 gggattacag gcacccgcca ccatgcctgg ctaatttttg tatttttagtagagacgagg 86280 tttcaccatt ttggccaggc tggtctcgaa ctcctgacct caggtgatccacctgccttc 86340 gcctcccaaa gtgctgggag tacaatactt ccagccaaag tacagtacttctattcaata 86400 gcaagctttg aagatttgct ttgtttctgc cagggaacaa tttttaagttatttgactga 86460 tcattgaaat caagggggtg gtattcccca tgtcacttta aaaagcttaatattattata 86520 atagttttta cttttttggc caggtggggt ggctcacgcc tataatcccagcactttggg 86580 aggcgaggtg ggcggatcac gaggtcagga gatcgagact atcctggctaacatggtgaa 86640 accccttctc taccaaaaat acaaaaatta gctgggcgtg gtggcaggcacctgtaatcc 86700 cagctactca ggaggctgag gcaggagaat tgcttgaacc caggaggcggagcttgcagt 86760 gagccaaggt ggtgccactg cactccagcc cggcaacaga gctagactccgcctcaaaaa 86820 agaaaaaaat agttttcact ttgtttacat ttatattgga atatataaatataaaggcat 86880 gacggttact ttgtcatact tttggaaagg catcaaagac aacttcagagtatactagat 86940 agtatactaa ataggtaaat catgtttaaa gttttttagt agtttaatctgtcagttttc 87000 cttttttggg cagtgaaact gatcattgca aactatctga aaaacacttttatctttgta 87060 ggtattcaat aatagccatt ctaaaaagga catatgttgt tgtccatcaaacatttccta 87120 gtctttactc attctgggca ttttctttgg tgctgattct ttattagaatcagattcaaa 87180 ttggaatagc tttttgtttt gttttccttt ttactacact ttcctcaaacatcaagttga 87240 tggtttcaga tacttccttt ttcttctgac agttatcttt ctaatttactagtccaatat 87300 ttaatgttac tgaaaggatt ttctgttttt cttttaattt tgaaagtttttagcatcata 87360 caaaagaggc ttattggaca ttttatgaat acctaattgg tgtgtatttgttgttatgga 87420 tagcagtggt gatctctttt cagattgcct cctcttccca acctaacctcagttaataaa 87480 ggtctgctct cctacctttt aaaattaact ttgtactgga gatatagaattactgtctcc 87540 agaattcctt taatttgaga ttgggattgt gtatatttta gtgtaatactgggatgttta 87600 atatggggcc ttcacctttt tgaggatttg ggaaccagtt atttgtcctgatcttcctcc 87660 tcaatttttt acagtagcac taaataaacc agaaatattt ttgggaaaatgatgtgcttt 87720 cctctcatga taaactcttg ctgttatttt ttagaaattt agaaaatgatttaagattta 87780 aattagccat tttacaggtc ttaggaaatg taataagagt ttgggaatacctatattcat 87840 cattggcatg cttattcttt gtgaggataa atggaaatat tttatttttttctctcccta 87900 gaccagagga agagtcttca tcatcctcca gtgatgaaga tgaggatgataggaaacaga 87960 ttgatgagct actaggcaaa gttgtatgtg tagattacat tagtttggataaaaagaaag 88020 cactgtggtt tcctgcattg gtgagtagct tcagtataca agagtttaatttmaaacttt 88080 ataagtttat gaagaaaaca attttcttac taatgttttt cataggtacaaaatgaggaa 88140 atgtttttag cattatttag ttcacactaa gcttactttc aacgttgtctttgaatacaa 88200 agaactcttg atgtcattca ggaaaggcaa aatcttgata atcttagaggtttagctatg 88260 tggaagttga attctctctc tctcggtaga aaattcatga tgcaaacttttaaagctcct 88320 aagtccatgt aggtctagat tgcaaaagaa caacagaatc tttcttggttgacagggata 88380 actagacctt ttcctcatac ttagtgaatc agggtgtttc catccttgatcttaatcttg 88440 ccttttaggt actccctata taatcatgac tgcaattata cattaccccactcttcaatt 88500 ttctctacag tagattgtta attctaccta ctttttaatg tttttctctccaaaagattg 88560 caattcctta gattccaggt tgaatatttt gcattttttc cttgttttctcacgttgcct 88620 ttttgtgatt ctgaggaggt actcaagatt tattatcagg agtatcctcatgattgtccc 88680 aagttcttgt atttcaaact gttaagacat gacagaaagc cttttgagctttttatctct 88740 ctgtttttcg taaccactag cttctttagc aagttgaaag ggccatgtatagaccatttt 88800 ctttgacttt tagaaaggga acatatacta atatctttca cggacataactaggctgctt 88860 ttcaaataat cttttggtag acaaaataat ttgtagctct taaagcaaatagaggttcaa 88920 ctaataatta gctatatatg atactttaat gaagatttaa ctctgtggcaataagaatgc 88980 atttttttct ggacagcata taattggata aatcacttaa ggttttaaatttcaagtagg 89040 agactgaagt taataatcag tatctcttaa gaattatctt ggctggggtgtggtggctca 89100 cacctgtaat cctagcactt tgggaggccg aggtgggcaa atcccctgaggtcaggagtt 89160 cgagaccagc ctggccaaca gggcaaaacc ccgtctctac taaaaatacaaaaaaattag 89220 ctggctgtgg tggcacatgc ctgtaatccc agctaggcag gagaatagcttgaacccagg 89280 aggcggaggt tgcagtgagc caagatcgcg ccactgcact ccagtgtgggcgacagagca 89340 agattccctc tcaaaaaaaa aaaaaagaag aaaaaaaaag aattatcttgaactctttaa 89400 aattttgata tgtctgaatt gtcctatgat gattttatct gaagaccattgagtcatttg 89460 catcaaacag tgagatccac tttttttttt tttccccgga gacagaggcttactccgtta 89520 cccaggttag agtgcagtgg tgtgatcctg actcgctgca gccttgacctcctgggctca 89580 acaattctcc cacctcagcc tcctgagtag ctcacactac aggtggagaccaccacatct 89640 ggctaatttt taaatacttt gtaaatacga ggtctcccta tgctgcccaggctagtctta 89700 aactcctgga ttcaagtgac cttcccacct tggcttccca gagtgctgggattacaggtg 89760 ggggctactg agcccagccc acatctttta caaatatgtc ctcccctcacttcatattta 89820 ataagctata aactgaagat ccgctgataa acaaaagaca ctttgaaagtactattctca 89880 cgtttgacaa ctaatttgtt ctcaaatatc gagagacaga atgggtcaaaattattctct 89940 gtctctgttt tgtatgttct cactccctta ctaaaaataa ttagccaaattctcctctaa 90000 tgcttttttc ccttatgtta atgacattca ataaaatttt tgtgttctcaagattaacac 90060 atatttgtta ggtatgcatt tatcatatac caagttctgt tcaaaatatttaacaaagat 90120 agactcattt agtggtgtag aggttttttg ttctcatgat taataaactttccttttttc 90180 ttttcttttc ttttcttttt tttttttttg agatggagtt tcgcccttgttgcccaggct 90240 ggagtgcaat ggtgcgatct cggctcactg caactccgcc tcccaggttcaagtgatcct 90300 cctgcctcag cctcccgagt agctggtatg acaagccacc atgcccggctaattttgtat 90360 ttttagtaga gacagcgttt ctccatgtcg gtcaggctgg tctccaactcctgacctcag 90420 gtgatctgcc cgcctcagcc tcccaaaatg ttgggattac aggcatgagccagcacaccc 90480 ggtgatgatt aataaacttt tctctacaat ttgtgcttga gaatctgctaactctttatt 90540 ctcattctct cattttcctg ctcattgact aacaattgca gagacttagaaatgggatta 90600 aaacactgtc ctttgtttat atgtatttgt atgaaaactt atgtccataatatatataaa 90660 ctcttttgta tatttgtggt ttttgtgcta aactaggtaa ccatggtctcaaatgaaaaa 90720 gaaacaatag aactaccttt gtttttactg ttttctactt gtgatgtccatttacccttt 90780 ttagtcaaca tgtcttattt ctttatctcc actgcttagt tcagtgttcggcacaaatta 90840 artatgcagt aaatgtcagc tcttgtaatg atagtgaaaa atgaggacaattatttgttt 90900 taataaatat aaatatttta taataccttt gtaccttcct gtgtatccaacatttagtgg 90960 atacctatat gtacaaggag tgtgctaaca cctattatga gataggtattattatttatc 91020 ccgatgtaga gataaggaaa ctgagtatta gcagggttaa gttagttgccacactttggg 91080 ttaattatta agctgaaaaa aagcatcagt gtttgcgatt ctttctttctttctaaagca 91140 ctaggagtct ttgtgagttt cttttgaaag tatgttttaa ggccagctgcagtggctcac 91200 acctgcagtc ccagcacttt gggaggccga ggcaggcgga tcacctgaggtcaggagttc 91260 aagaccagcc tggccaacat agtgaaaccc cttctgtact aaaaatggaaaaatttgcag 91320 gacatggtgg tgcgtgctta tagtcccagc tactcgggag gctgaggcaggagaatcgct 91380 tgaacctggg aggcggaggc tgcagtgatc tgagatcaca ccactgcattccagcctggg 91440 tgacagagca agacttttat ctcaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaatatata 91500 tatatatata tatatatatt ttttataaaa ccgttttgtg ggccaggcgcggcggctcac 91560 acctgtaatc ccagcacttt gggaggccga ggtgggcaga tcacttgaggtcaggagttc 91620 aagaccagcc tggccaacat ggtgaaaccc tatctttact aaaatacaaaaattagccga 91680 gcatggtggc gggagcctat aatcccagct acttgggagg ctgaggcaggagaattgctt 91740 gaacacggga ggtggaggtt gcagtgagct gagaccgtgc cacagcactccagcctgagt 91800 gacagagcaa gaccccatct caaacacaca cacacacaca cacacacacacacacaactg 91860 ttttgtaagt ctttttctca ataggaacat taaatttttt aatattagtgttttgagtta 91920 ttaatttaag cttctcacac cataatcact aaaaagtttc tttgacgaaaatgaaaaatc 91980 taaggatttt taatattgaa aggttagtaa tctgtttagt cagccatttcactgttgttt 92040 ttaaattctt agaattattg atgtatgtta tctcctccca aaattgagatccattggacg 92100 gtgatggata gtttttggta actagagtga aaaattatgt tctaaatcgaccaatcttga 92160 tataatcgca caaatagtat ttgtgtttct aatttatgtt atttttataaatcgagtaaa 92220 ttcaaagtgt tttagtaaca tcataagaaa acagttaact gggtctgggtggcctgtgcc 92280 tgtagtccca gctacttggg agactgaggt gggaggatcg attgaaccctgggaggttga 92340 ggctgcattg agtcgtgatc acgccactgc acagagtgag accttgtctcaaaaaaaaaa 92400 aaagaaaaag aaaaaggaaa caactacata gtcccaggaa ctataaataactaccatgca 92460 ctgagtattt accctgacta ggactgtgct gagcttatta atataagtagtattgtttaa 92520 tcctaattga gacttactct cagtctcaat tttttatttt ccaatttcattttaagaggg 92580 ggaaatctta gattccatgg ctcatgcagt tctccttttc agtttttttcaccaggttct 92640 cctcttctaa tcaccctttc aagctgtaat tcaggattca gaatgtatcgctgtattttc 92700 tttacggtga tctcatctcc ttttaaagct tcaccttgca gagatagcaaagagataaag 92760 tgaccaagaa aataacatac atgggtgaat gggataaaat gcatattgtcgttctgtggc 92820 agaactggac agtgagccca cttactcata caccagtgtc atcactggccaaagctatca 92880 catgtgcagt tttcgaaatc cttcgctcat cagggaaaag taatgagtggatttggctca 92940 caagccccaa gttttagacc cctaatattt aggctgtggg ctctcaaatttatatccttg 93000 agtccagacc tctacgcttt gctccagacc tgatttttct gttcactgtcttcatttgga 93060 tgtcttttag ccacctaaaa cttagtagtc ccaaaactcc tcttattatcccttccctca 93120 tgcacaaact tggccctttt ttgttgtttt tctttcacag tgaataactctgtcatctgc 93180 ctatttgtac aaggcagaaa actaagcatc ctcttattgt tcatctttctacttccctaa 93240 ttatcttgga tatttctcat tcctttaatt tgccattgcc accactttacatccaagcca 93300 ccacctctca ctcaggtgat cacagtagct tagtttcttt gaacagttgcctctgcgtgg 93360 tccattttct tcatagcagc caaagtagat ttttaaaagg aaaatctattatgacattct 93420 ttggtttcag attctttcaa caaagatgtt agattttatc ctaagaattaatatggccta 93480 caaggctctg gaaattctgg acccttgttt ttttcttagc catgatgtatcccttcagtt 93540 aaggatctaa ctctaccttt tttctgtttc tgactagaac ttcgtccacagcttcatcct 93600 agtcatagct gtgcttctgc tttcaatgcc ttgcctatgc caaaaattctagattatcca 93660 gcagctggaa ggatgtagga tgccaattta aaattgcttg tgtgtgctcccacgtgtgtg 93720 tgtgtgcctg tttaccctca aactaaaatt agtttttaaa cttcaagcttacacttttta 93780 aaaggattta aatggtgggt acttacctat ctatgagttc ttttagttataatagcttca 93840 ccctttttct taaaaggtgg tttgtcctga ttgtagtgat gagattgctgtaaaaaagga 93900 caatattctt gttcgatctt tcaaagatgg aaaattgtaa gtataatttacttggtttga 93960 aaaaccagtt tataaatata tgctgttatt ttgaattagg atgttaaatgattaattagt 94020 tatttccaat taccttaggc ttttattttt catcatacag aatgtttcaaagagtcactg 94080 tttttcatat tatcttaatg tgttgtagtc ttatagtatg tttttccttactaattttgg 94140 taatcttttc caaactagac actcagtact gtctaatttg tcaacacttgtccacataat 94200 agctggttat ttgaggcaaa tttgccatat ctaatgtatt taatactttattgatttttt 94260 tgtttttttt gagacggagc tcactctgtc acccaggctg gagtgcagtggtgcagtttc 94320 ggctcactgc aacctctgcc tcctgggttc aagctattct cctgcctcagcctcctgagt 94380 agctgggact ataggtgcat gccaccgcac ccagctaatt tttatatttttaatagagac 94440 ggggattcac tgtgttgtcc aggctggtct cgaactcctg atctcgtgatctgcccgcct 94500 cgaaccccaa agtgctggga ttataggcgt gagccatcgc gcccggcctattgatgtatt 94560 tattgacatt atttccttag ggtttgttct tgatggtcta attaggaatttccaatactt 94620 ctactagtta tgtctaatgg ttatagtgat acctgttgga taatactcaacttttgcagt 94680 agtctgagga ttaaaatggg ttacttgtat atggatggta agtgttaagaaaatgatttg 94740 ttaatttttc tttaagaatt atatgactag agaagccaga tattttatacagaattgtat 94800 tgggtataaa taccttaaaa aggtcatcat gttttaagat ttgatgtttgaattttcaag 94860 ttcgggaaag gtatttttat ttttaattat tttattttac tttatttcatttttttggtg 94920 agggtctcac tctttcaccc aggctggagt gcagtgacat gatcatggctcactgcagcc 94980 atgacccttt gggctcaggt tatcctttca cctcaatttc tcttgagtagctgggactac 95040 acctggctat ttttgtattt tttgtagtga taaggtttca ccatgttggccaggctggtc 95100 tcgaactcct cagctcaagc gatccaaatg ccctggcctc ccaaaatgctgagattacag 95160 gcgtgagcct ctgcacccag tctgaaaagt tagcttcaaa aacagtatgtaggctgggcg 95220 cggtggctca cgcctgtaat cccagcactt tgggaggcct aggcaggtgggatcatgagg 95280 tcaggagtgc aagaccagcc tggctaacat ggcaaaactc cgtctctactaaaaatacaa 95340 aaattagcca ggcatggtgg tgggcgcctg taattccagc tactagggaggctgaggcag 95400 gagaatcgct tgaacctggg agatggaggg tgccgtgagc agaaataatgccattgcact 95460 ccagcctggg tgacaacaac aagattccat ctcaaaacaa aacaaaacaaaacaaaaaaa 95520 aatgtagaac caactttaac aaaattggga taatcagagg aatttgagcacagaaattga 95580 atattgactt aatattaaat tgttgggggg taaggaaaat gggaaaacgttagtaaaacg 95640 gcacaaactt ttattggtaa gatgaataaa atgtacagga tctgatgtacagtgtggtaa 95700 ctagtattct ggcagcctcc caagtagcta gcactacagg tgtgtgccaccacgctcggc 95760 aaatttttgt agggtttttt ttttttgcca tgttgcccag gctggtcttgaactcctgag 95820 ttcaagtgat ctgcccactt tggcccccaa agtgttggga ttacagttgtcagccactgt 95880 gtccaacctt tatttattta tttattttat tttatttact tttttttgagatggagtttc 95940 actcttgttg cccaggctgg agtgcaatgg cgcaatctca gctcactgcaacctccgcct 96000 cccaggttca agcgattctc ctgcctcagc ctctcgagta gctgggactacaggcatgca 96060 ccaccacacc cagctaattt ttatattttt aatagagatg gggtttcaccatgttggtca 96120 ggctggtctt gaactcctga ccccaggtga tccactcgcc tcagcttcccaaagtgctgg 96180 gattacagtt gtgaaccact gcactcagct caacctttat tttttaagagatgggatctt 96240 gctctgttgc ctacgctaga gtgcagtggc acgatcatag ctcactgtaaccctgccctg 96300 ggttttttcc agtcagctcc ctgcctcaga ctcccaagta gctgggactacaggcacatg 96360 ccaccactcc tggctaattc tttgcttact tttttagttt gtctgtggagactgggtctc 96420 gctatattac ctatcctggt cttgaacttt ggcatgaagc agtcctcccaccttgacctc 96480 ccaaagtgtt gggattacag gcatgaatca ccatggctgg ccccaaacattttatcatta 96540 tattgccata aatcgttttt ggccgggtgt ggtggctcat gcctataatcccagtacttt 96600 gggagactga ggtgggtgga tcagttgagg ttaggagttc aagaccaacctgtccaacat 96660 ggtgaaaccc tgtctctact aaaaatacaa aaattagccg ggtggtagtggcatgtgcct 96720 gtaatcccag ctactcggga ggttgaggca gaagaatcgc ttgagcctgggaggtagagg 96780 ttgtggtgag ccaagattgt gccactgcac tccagtctgg atgacagagtgagaccctgt 96840 ctcaaaaaca aaacaaaaca aaacaaaaaa agttttcagt gttgcatttgggcaggggac 96900 tctggtattt gggaacacgt tggtgtttat aaaatagctc ttatggtaggatccttctgt 96960 agatgattac gtattttaac atcctgtctt gtatgttatt tctgacttgctactctgtgt 97020 gcaatcactt ttattgtaaa cgtcaacatt cccatatttt cagtatctttctgtgatatg 97080 taatcatctg gattcttaat taatctcagt catattttgg gktttttttcttctcttata 97140 attaaaaaaa atatatagta cttcagttcc aagaaaagat gtccatgaaattactagtga 97200 cactgcacca aagcctgatg ctgttttaaa gcaaggtaag gataatcttggaataaatta 97260 caagtactgt aaaaactgct aaagtgtttt tgaagattaa atttttctttgctctttaaa 97320 atttttcttt aaattatgtt tttatggtta tatttggttc cttgtgacctttatttttat 97380 aaacccatgt gtcttctgtg ttataagttt ccttttcttt tttttttttggagacagagt 97440 cttgctctgt cgcccagggt ggagtggcgc aatctcggct cactgcaacctctgcctcct 97500 gggttcaagc aattctcttg cctcagcctc ccgagtaact gggactacaggcacacgccg 97560 ccatgcccgg ctaatttttt gttattttaa tagagctggg gtttcaccgtgttgcccagg 97620 ccggtctcga actcctgagc tcaggcagtc cacctgcctc agcctcccgaagtgctagga 97680 ttacaggtgt gagccactgc gcccaaccat aggtttcctt tttttttctgtttttttttt 97740 tttttttttt tttagcttat agtcagtcat aataatctag aataatgggagagaactgtg 97800 tagtcagctg ttactataat ctagcttttg cctggtaaat aaaatattagtttctgggaa 97860 tggtagaaga aaggaatcaa agtaactttg tgttgttgtt ttttttgttttgttttgttt 97920 tgttttgttt tgtttttttg agatggagtc ttgctctgtc gcccaggttgcagtgcagtg 97980 gtacaatctc ggtgcaccct ccacctcccg ggttcaagcg attgtcctgcctcagtctcc 98040 caagtagctg ggactatagg ggcccaccac tatgcccggc taatttttgtgtttttagta 98100 gagacagagt ttcaccattg accaggctgg tatcaaactc ctgagctcaggcagtccacc 98160 cgccttggcc tcccaagtgc tgggattaca ggcataagcc atcacgcctgactgaggaat 98220 caaagtaatt ttggtttggt atttctcact aatgaatgta catccttaaccacaaggctc 98280 cagtagtttt atttgaggaa tatgtggtaa ttgcatctgt cacttgatttttggcactgt 98340 aaatagttgt cttctctttg cccttattcc ttgaattcag taatatgctatgtagattgg 98400 ttaaaatcat caagattttt tggtatataa tttattccca tatattataaaatgagaata 98460 attgtgtcta gctaatttta gttgaaggta attgttacca tagttttaattgaagttcaa 98520 ctgaaaatgt aaaaatcatg tgtggtcagt tttcaggtat tgtatatttattttaatggc 98580 tctgtgcctg gtattaatac tgtttgataa ttgtttgttg cataagatattacatgattt 98640 cctgtattaa aggtttttac tagtgaagct taatctttgt ttaaaagaataaaactgctg 98700 ttcttttatt tatttaaact taagtaatct tccaatggtg gcagttttgccatctataaa 98760 atgactacct aatgcatagg atggatagga ttaaatatat tgctacatttaaaagcactt 98820 aacacagttt ctggtacata gcaaacaata aatgtcagtg gtattagcacagtagttttt 98880 atttctttat cgtgtttaat actttttgag aaaacaattg tcttaatttcttcagttcat 98940 gtaccttgtg gtctgtttcc agcctttgaa caggcacttg aatttcacaaaagtagaact 99000 attcctgcta actggaagac tgaattgaaa gaagatagct ctagcagtgaagcagaggaa 99060 gaagaggagg aggaagatga tgaaaaagaa aaggaggrta atagcagtgaagaagargta 99120 agtgaaaaca gttgatacct tttaaaatta taaataacag ttgggtttcccttgtgggtt 99180 aggatttggc attaagtcat tactcatata agtattttta gtatgagaaatatttcataa 99240 tcttgtattt gagatcctca taatcagata gtttacttgg ttactttagatatatgatct 99300 tgtgaaatag aaaatattaa gcctgatttt cctaataacc tcaaaaacatgtcttagtct 99360 tcctatgctt tttttaatga gatgattaag aaagtatcca ccaggctgggcgcagtggct 99420 caggcctgta atcccagcac tttgggaggc caaggcaggc ggatcacgaggtcaggagat 99480 cgagaccatc ctggctaaca cagcgaaacc ccgtctttac taaaaatacaaaaaattagc 99540 cgggggtggt ggtgggcacc tgtagtccca gctactcggt aggctgaggcaggagaatgg 99600 catgaaaccg agagacggag cttgcagtga gccaagatcg cgccactgcactccagcctg 99660 ggcgacagag cgagactctg tctcaaaaaa aaaaaaaaac gagaacaaaaacaaaaaaac 99720 ttaccatcta ggccaggcac ggtgactcac gaccataatc gtagcactttgggagggcaa 99780 ggcaggtggc taacttgaac tcaggagttt gagaccagac tgggcaacatggtgaaaccc 99840 catctcaaca aaaataaaaa taaaaataat tagcagggca tgtcagtgcactccagcccc 99900 agtgcactca gtagccccag ctactgagga ggctgtgttg ggaggacggcctgagccctg 99960 gaggtggagg ttgcaatgag ccgtgaattt accactgccc tacatcctgggtgacagagt 100020 gagaccctgt cttattaaaa aaaaaaaaaa aaaaaaaaaa agaacctacctacctaattc 100080 tttaaaatgc ttttcattac catgttgaca gctgtcacct catgaaatgcttacatctcc 100140 atattttatt gattaaccca gaaaagtata aaatacatct ttcatttaatatttaaaaga 100200 aaaatcaggc cgggtactgt ggctcatgct gggatgacta atcctagcactctgtgaggc 100260 cgaggcgggc agatcatgag atcaggagtt cgaggccagc ttggctaacatggtgaaacc 100320 ccgtctctta ctaaaaatac aaaataatta gccaggtgtg gtggtggacacctgtaatcc 100380 cagctactcg ggaggctgag gcagtagaat tgcttgaacc cgggaggcggacttgcagtg 100440 agctgagatt gcgccattgt tctccagccc gggtgacagt aggagaccccgtctcaaaaa 100500 aaaaaaaaaa aaaaatagca gaaaagtaaa gatcaatatc tctttgattcagagaatctt 100560 gaatcatttt atctaagaca aatgggtaga tagttctgat tagatcataaataagccata 100620 aaatatggct caaagagctc aaattttgga ttttatttta cctggagctttagatctggc 100680 agtgacaagg tactctttaa tggtttgctg aacagcctcg taaaagttaatgagtggtgc 100740 agatagtgaa ggctgctata tttgaccatg aaatgaaatg tatttaatgtgtttagtaaa 100800 ttaattggtc agtatgtaat actttaggta agttatgaaa ttctgccttttaactgtagt 100860 aaagtattaa tgaaatgaga ttgttttgga ggttatattt tagggtgtagtgatctgtaa 100920 tgacaatgta taatgtagaa atttccctgc ttcagtcttc tccctgactgctttctattt 100980 aaattgctaa aatcagcaat cattcccttg ctgctaaaaa cctcctgagtagctttttgt 101040 cactgtaaaa gttaagcttt catatagaat ctcctttata gtccaattcctgcctcaatt 101100 ttttctcatc atttcccttt ctgtgtcctc ccccttgaac tttatgctatagcaatgtca 101160 aattacatat gttgctgcta ctcacctttg catatgctag tacctctgtaggcaccacct 101220 tttcctcccc tccttcctta gtcagcatgg tacattcctg ttcaattttcagaaccctac 101280 ttaggtgcca tctctctaaa gagaaacctg tgaaccatca cctccctcacctcaggaaag 101340 caccagttca tttgctagtc taaactacta ctgctcctgg caggcacggtgggtcatgcc 101400 tgtaatccca gcactttggg aagctgacgc tagcggatca cctgaggtcaggagtttgag 101460 accagtctgg ccaacatggt gaaacccgtc tgtactaaaa atacaaaaattagctgggtg 101520 tggctgggcg cggtggctca cgcctgtaat cccagcactt tgggaggccaaggcaggcag 101580 atcacctgag gtcaggagat cgagaccatc ctggctaaca tggtgaaaccctgtctctac 101640 taaaaacacc aaaaaattag ccgggcatgg tggtggacgc ctgtagtcccagctactcag 101700 gaggctgagg caggagaatg gtgtcaaccc gggaggcgga gcttgcagtgagccgagatc 101760 acgccactgc actccagcct gggtgacaga gccagactcc atctaaaaaaaaaaaaatta 101820 gctgggcgtg gtggcaggtg cctgttatcc cagctactag ggaggctgaggcagcaggat 101880 tgcttgaacc caggaagcgg aggttgcagt gagccgagat gacaccactgcactccagcc 101940 tgggcgacag agcaagactc tgtctcaaaa acaagacaaa acaaaaaaagaattactact 102000 gctccttata catatttttg tcatttatat ttattgtcta ttttttcttccccacgaatt 102060 tgtaagttcc ttaaagttag gaatgctgtc ttactcgtat tcatacttaaagtgtctgtc 102120 atagtgtgta aaatataaca gatgataaat agctaccaaa ccttttggtgcttagtgttt 102180 tcacaagtgt ttttgtaaaa gcaaatctgt agtgtagtgt aaacattttcttgagttaaa 102240 agcttacctt aaatgttttt ggaataggaa aaattgtgct gtttattaaagttaaatatt 102300 gtaccatggg aacatttaag ataacatcta gaaaatagat gtccttagatttgtatttga 102360 tcaccttacc aaaacctgaa tgtatggagt gtgtttaaat tgagaaatacagattcatca 102420 ttagcaaatt aatggttgtc atttattttt ctctgcccaa ttagaaaacctaatagttga 102480 tcaacatagg ctttaaaaaa aaaattacct ttctttgttg tactagcttttaaattaaaa 102540 agacaatctc gctaaagcag tggcattagg cacatgattt tccaaattgaaaaatgctat 102600 gtttttattt atttagatta ccagttgaat atcctaactt ttactgttacaaatctgtat 102660 atttaagcca gagtaactgt aaagcctagc tgtatatttg gaatcattttctctgcagtt 102720 actcctaatc atgttctgta acagtaggct ccatagccta ttttttcttttatttgattc 102780 agcacctggc atagtaccta cacttacagg tgctcaagtg tttgttgaatggatgaaagt 102840 cattgaattg gtgagttcct caatcttaac ctactaccct gctccttgcaggttgcttca 102900 ggaccagctc cctgctgcca cagcccttgg ccacagcagc aagaagctagtccttccctt 102960 gtaattgata aattggggaa ctattttatt acttgaagat ttccctaagtctgaatcttc 103020 aggtattgta gatcaaattt gttccccact ttactgatta tgtaagcttgaagtatatac 103080 agtggggctg cataaaatga aggatacttg cagtcagcta atgggacccatacttttata 103140 gtaccttgag gagcaataat agaattagtt ttgatgttta actctgatccagtttcgcat 103200 aagttgagag taggtattct tgaacctgtg atcctgattt gaaaaatagctctctcatat 103260 ggtaaaaaaa acaaaacaaa acaaaacaaa accacgaaca gtcttgctagtcccttttct 103320 catatgggaa tttttactgt ggggattcta actattggga tactttttaaggcatattcc 103380 tctataaaac ataaaatgtc tagacttacc tggttttgaa cagcttagtgttaaaagagt 103440 aactttgatt acgtaaaaag cctttgaagt attttaatga acactagtctttgctattgg 103500 taagaaatct gcttgtttta ttaaaatgct taattgaaga aaataatattcttctgtgat 103560 taaaattagg aagaaataga accatttcca gaagaaaggg agaactttcttcagcaattg 103620 tacaaattta tggaagatag aggtgagtat tttttattta tcattaacgtggtaagtttt 103680 ggacagataa ttagtatctt aaaaataaga atagaatttt gtttactgaactttatgtca 103740 tcagtaatta tggctgtctt tttgactctg atatcaccag cacctagcatagcgcctgtc 103800 acgtawcaag tagaatgagg aatttgatag cattttgaca gatattgtggctatggacac 103860 ataaatgcac ttattaccct gtcctgccaa tccctttacc tatccatgggtccctaaaga 103920 acagtagcaa aatgggaatt aagccagaaa tagataaaga agtgtgacaaagtctcaaaa 103980 gtgagccttg gctcagctta tttaatctgc cagaagctgc taaggccacggaaggattga 104040 aggaagaaga ataacttagg gtaatgaact aatccttccg taacctcaacactgttattc 104100 tctagcttat cttcttggtc agagtctact ttcttctttt ctgggaagatgcttgtaagg 104160 gagtaaggag aggctttcta gctggaaggg aagggcaaaa tgaatgattagtgtttggaa 104220 acaccacaat actctgggaa ttgaacgttt tctaaatccc tgattgtaatgctgatgagt 104280 agaaactgac tctgtttgtc atgtttagtt ttcataaatg ataacaacgcctaacacaat 104340 ataagtatat tttctttttc aatataaaat gtctttgatt cttgtattatttctttacat 104400 gaatggaggg ttggcctaag aagtatgctg tttcataggc tcttgtataagataaatgaa 104460 aaagcaaaga aaatgaataa aatctaatac tagaaaagaa tgaaaaaatatgattaagcc 104520 agtcatatat tttgtagaaa tggccatatg aggcattgta cccctaccctgttacctcct 104580 ctaccacttt ctcatggttt gtttgattta gcatccttgc cttgcgttttcttgtgttag 104640 aatgaaaaaa aaaaaaaaat gtagctttac tatagaatca ttatttagagtagtagtaaa 104700 ttggtaaaat acaaagaaaa tgccataaaa gtaactctta ttgggaagcagttatctagt 104760 atttcttccc tcagcagagg gaagaaagta gaatgaaaat aaaaggtttcaagaatctgg 104820 ttatggtttt taaaatttaa aaaattttaa agtggccatg ggcttatttatgtgtagata 104880 ccatgtcttt gattaacaag ataatttctc atatatatat atacatatatatgtatatat 104940 acacacacat atatatacgt gtatatatat atatacgtgt atatatatataaaatactat 105000 aaatacttgt attatttgaa tcttttgaaa ttttaaatta aaaatgtagtgtggatctaa 105060 tatagtttgt gttttctgta acaggtacac ctattaacaa acgacctgtacttggatatc 105120 gaaatttgaa tctctttaag ttattcagac ttgtacacaa acttggaggatttgataatg 105180 tgagtgttgt agtcaaactg aaacatcttt ttcatgtaac atgttttctcaggtatctgc 105240 catctctgaa attgtttttc taaacaacaa aacaaagttt ttcagtaaaaacctaagaag 105300 aagataaagt ggaaagtaca gctagcctcc tcagacaaaa tttaaattttaaagattttt 105360 taggccaggc atggtggctc atgcctgcaa ccccaacact ttgggaggctgaggcgggta 105420 gatcacttta ggccaggagt tcaagaacag cctggccaac atagcaaaaccccatctcta 105480 caaagaatac aaaaatcagc cagacgtggt gacgcacgct tgtaatcccagctacatggc 105540 aggctgaggc acgagaatcg cttgaaccca ggagacagag attacagtgaaccaagattg 105600 caccgctgga ctgcattcca gcctgcgtga cagaatgaga ctctgtctcaaaaaaaaaaa 105660 aaaaaagatt ttttttcata tcagctttct gattactgtt aaattggtcaatctttcata 105720 aaccttcttt ttttatctga gtcacagcac agccatattg gtggaaatccttcctgtgtc 105780 tctttttcat tagacagtat gctccttaag agcagggatc tgtcttaatattctgtatat 105840 tcttaatccc tttcagaata taaagggatt aaagcaggaa atgtttgttgaactgatctg 105900 aagaaattat tttaaggttg acgtgatgta cgatacttat ccaattgagaatattctatg 105960 gaataaaata ctttacaaat ttctgcagaa ctgcatttat tagcctgtatattttatgcg 106020 aaataataaa tcctttaaaa atgtttgtct ccatttcttt gttaaaaattttgatttaag 106080 agcctgaata tttttccatt ttccataact gggtttattt aaaagtcaaatggggccagt 106140 tatggtgact catgcctgta atctcagcac tttgggaggc taaggccaggggattgctgg 106200 agcccaggac tttgagaccc gcctgggcaa catggtgaga ctccatctccacaaaaaaaa 106260 ttttttaatt agccaggtgt ggtggtgtgc atctatagtc ccagctacttgggttgttga 106320 gtcaggaaga tcccttgagc ccggaagttc agggctgcag tgagctgtgtttgtgccact 106380 gttgctgcac tttagcctag gcaatagaat aagatcctgt ctccaaaaaaaaaaaaaaaa 106440 aaaaaaaaaa aaaaaaaata gtcaaatgac ttaatatttc aacatttataattttctgat 106500 atttgttaga gtactaaaac ctttattttt aaaccaaatt tagaatttatcaagttttta 106560 aaaattcttt tgagtctagg accaataaat ataaaaatat actcatatatactgtgtcta 106620 tcaaaatgtt attatattaa caccttttaa tttgagagtt tttgtttcactttgtccatt 106680 agattgaaag tggagctgtt tggaaacaag tctaccaaga tcttggaatccctgtcttaa 106740 attcagctgc aggatacaat gttaaatgtg cttataaaaa gtaagttagtataattgata 106800 tgattatctt cagtattttt acctagaaag caggatgaaa tttaccttataactgaggtt 106860 aattatttct ttaaagaaat cagaacattt tactagagta gtcattctatgatagtattt 106920 tctaaagtgt attctatgat actagatcta tgagaaattc tgtgaagaaagtttttgtgg 106980 ctaagtacat ttgtcaagta cttcatagag gttaaaaagc taatattctgaagtcaagta 107040 aatccacatt tgaatcctag ttctgccaat tattaactgt ttatgccctagggccagtta 107100 tttattctcc taagcctcag tttcttcatt tgtaaaatgg aatgcttggccaggcacggt 107160 ggctcatgcc tgtaacttca gcactttggg aggccgaggc gggcagatcacttgaggtca 107220 ggagttcaag accagcctgg ccagcatggc gaaaccttgt ctccactaaaaatacaaaaa 107280 ttacctgagc atagggctaa cgcctgtagt cccagctact caggaggctgaggcaggaga 107340 attgcttgaa cccaggaggc ggaggttata gtgagctgag attgcgccactgcacttcag 107400 cctgggcaac agagtaaggc tgtctcaaaa aaacaaaaaa aaacaaaaaaaaacaaaaaa 107460 aggatgctgc tgtgtgcccc agggttactg tagtgactaa atgagatttactaaggagtc 107520 cagcctcaca cataagaaat gtgcaaaaaa tagatgctat tactaatacttttaatagct 107580 gttattgtta tgactctgag cacagtgttt taaatgaatt ttttgacaaaattttttttt 107640 tgcattgaaa agattagaaa gaactagaat aaattagaat gttaatatggctcaatatct 107700 gagagtttag acaaaagaaa tgaatacaca ttaaaaataa ctctatagttatattttgaa 107760 atgttttcac caaatgtaat ttctcttcat gtttccagat acttatatggttttgaggag 107820 tactgtagat cagccaacat tgaatttcag atggcattgc cagagaaagttgttaacaag 107880 caatgtaagg agtgtgaaaa tgtaaaagaa ataaaagtta aggaggaaaatgaaacagag 107940 atcaaagaaa taaagatgga ggaggagagg aatataatac caagagaagaaaagcctatt 108000 gaggatgaaa ttgaaagaaa agaaaatatt aagccctctc tggtaaatcagatattgtta 108060 attgtctttt gctttctttg gtatattttc catatcctct ataaagttccaaaatcaata 108120 tattgtataa tattattctt tattatttgt ttattttctt cattaagtgctacttttgta 108180 taatttttat cataacttca aaatagctgt tagctatttt tgtctagagctttaaagtag 108240 caggagtttc ttaaaagtaa tttataactt ttaatattag aattggttgatacctcctgt 108300 tgctaagrga taaaccatgg atataggttg aaaattttta cttttgttggtttctttcct 108360 ctgatctttt tctagggaag taaaaagaat ttattagaat ctatacctacacattctgat 108420 caggaaaaag aagttaacat taaaaaacca gaagacaatg aaaatctggaygacaaagat 108480 gatgacacaa ctagggtaga tgaatccctc aacataaagg tagaagctgaggaagaaaaa 108540 gcaaaatctg ggtaagaaaa ttgtttttgg ttacgatgca tttcagcttgatggattctt 108600 atcatttgct ctttgtatgt gtttatgtat gaatctaaat taattgacaaaatattaaat 108660 aatagagtga ttggcattta ttaattaaat agcattcact tctttcagagaccctactga 108720 tagaaatttg tagagaaaga tcatactttg atccaccctt cttgctctcatcatggaaat 108780 tgataaaatt tttagctaga gcaaagtact ttttaggtct gctgaattacttgataatat 108840 aaacttgggg ctatgtccat gttttatata ttgtatgttg gctatagattctgagagttg 108900 tatttatttc ttaagcaaaa gcaagagtgg aaaaatacta aagttttaaaaggccaaaaa 108960 ttgtatgttc tattttatgt gatagtagca tttgtttgtt ttagtcacttggaatgatga 109020 attatttgac aatgtaattt atgcactgct agtggacccc tggattttatagataatctt 109080 tggcattgaa tcttttttct gaggcttaaa aaacataaac atatctgttagtgtctcatt 109140 taaataagta caatgaatta aaaaaaaaat catttagcag gccaggtgcggtggttcacg 109200 cctgtaatcc cagcactttt gggagaccgg ggcgggcaga tcacttgatgtcgggatttc 109260 aagaccagcc tggccaacat accgaaaccc tgtctcttct aaaaatacaaagaagtcagg 109320 cgtggtgaca tgtgcccata atcccagcta ctcaggaggg tgaggcaggagaatctcttg 109380 aaccctggag gcagaggttg cagtgagctg agatcgtacc actgtaggccagcttggggg 109440 atcgagtgag actctgtctc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaatca 109500 tttagcgtac tgacatttgg ccacagtatg cagacgaaca gggaaggggaaaataaaaaa 109560 tggggcttat atgaatatag taaaaaatgt ttattatact ttgttgtatttttgtgtata 109620 aaaatatata tttgaagtat gccttactgc tgttggagac tatttcaagtattaagaaac 109680 agtaaagatt tcagcttttt gcctaaaatc ttttgattga tcagagttctgaaattttgg 109740 tggcaccata caaacatgga agaaaaacat cctgggttta gaagaatgtagtaaaagtgt 109800 ttgttgatta tactttgtta tatttctatg tgtagaagtg tatgtttaaagtactgctaa 109860 aggagatcat tttgataaga atcttaataa agattcagct ttttaccaaaaaatctgatt 109920 tttaaatctg cagatgtttc tggaacatct gctgtttgca aggcactgtgataaatctgt 109980 atttttttct ttgctaattt tttaagttgg tgcttctgct tattttatagaataaactgt 110040 attttgtagg tttttaacaa cccctttaac tgtataatta tatggatttttatgtactac 110100 agatatttat tagtatatgc gtacctcaaa ctaaaatttt cctgatgtgactgccctagt 110160 ttggtagata ttttttagaa gcattgcagt tgggactgtt tatatagtactccctgcttt 110220 tccacagttt cagttacctg tggtcaactg tggtctgaaa acaggtgagtacagtgcaat 110280 aaaatatttt gagagagtga gacaagacca tattcacata actgttattatagcatatta 110340 taattgcttt gttattagtc attgcttatc tcatgctgtg ccttattaataaattaaact 110400 ttatcagagg tatgtatgta taggaaaaaa cacagtatat ataaggtttggtactatcca 110460 cggtttcagg catccactgg ggatcttgga ctgtatccct tgcagataagaggggcttac 110520 tgtaattgat ttttgagttt tattagataa ttgcagtgga aaattttacagtgataatta 110580 atatataggt atttttctgg catataaact ttctctaagt aataggctactaccaagaat 110640 gttcagtgtc cttcttccat tgtccagatg tgggagattg aagaagttgatatatgtaaa 110700 atgtctagtt cagtgccttg gccatagcac gtacttaata cactatcttttttaaaattt 110760 ttgtttcatt gtaaagagtg taatatcaag tagggaagat ttgatatttctttgtgaatt 110820 cagatgaaaa tccttgctga tacatttcgt ttcccaagat attcttctctaaaatatgac 110880 tagcttaaat gcatatgtgt gtgtatgtat gtatgtatgt atgtgagtgtatgtatgtat 110940 atgagtgcat gtatgtagtg tatgcatcaa cttaattttt tttaacctgtatgtatgttg 111000 gttgcatttt atactttcag aggctctctt tcttggcatt ctgtgttccatctggtttag 111060 cacattagtc ttgaatttgc cactgaagtc ttagagagta gaatagttttctttggttga 111120 ttgccagtaa ttatgttgtt tttttcttta tacttcagtg tattattaaagtgaatttca 111180 tttggtttgt gtctgagggt gatattaaat gtatattttt aatttttgattgcttgattt 111240 cattaataaa agagtgttgg gtttttgttg tagattgaat gaaaatttgcctgggctact 111300 acttaaagta tgattgtgct tgctgttggc atttagaatt gtttgtattcatcaactcag 111360 tgtattctat gatgtaagat acagcttgga gtttgtcgtc atttcaaaaaatttagaatt 111420 ttgattctat atctcatttt tatatgatga ttctgaggtg catgataaatctcttttatt 111480 gtggccacaa taaaatatgc ttcttttccc cactatgcaa agatagagtcttatgactta 111540 ggcaagtatt ctttcaaaaa aaaattgttt taatttacta tataaaataaaattcctcat 111600 aataatttat cattgataat cccatttctg ttaatttttt tgatattttagccttaataa 111660 aattactttt ttataataga aatattattt tcaataacat tgttaatattttaatctcag 111720 gctgggtgcg gtggctcatg cctgtaatcc ctgcacttcg ggaggcccaggcaggcggat 111780 cacctgaggt caggagttcg agaccagcct ggccaacatg gtgaaaccccatctctacta 111840 aaaatacaaa aaatcagcag agcgtagtgg catgtagtcc cagcttattgggaagctgaa 111900 gcaggagaat tggttgaacc tgggagacgg aggttgcagt gagctgagatcacgccactg 111960 tactccagcc tgggcaacag agcgagactc tgtctcaaaa aaaaagtatatatatataca 112020 cgtatatatg tgtgtgtgtg tgtgtgtgtg tatacatata tatgtatatctcagattata 112080 gctggtttca tgaaggtttt agatttttta aatacttata tatttgactgtaactgagtc 112140 tttaaaatag gaataaatct tgagaatttt tgaggaattt gtgttatagttcagacaaaa 112200 ttgatatatt tgatactttc gtgaacacgg acttacttcc accggtccattaaacatcct 112260 gccaacactc ctgccccact atacaaaagg acaaggagaa taaaaaagcattatagtgct 112320 gcttatatca ctatttatat attatttttg ttgcctgtat aattaattaggaatgataaa 112380 gtatattatc cctaaaaagg ttgcatatag agatataaag ctgaaattttcagtggagga 112440 agtttttttt ttaatataaa tattatattc atcaggtggg cacagtggctcacacctata 112500 atcccagcac tttggaaggc caaagtatga agattgcttg agcccgggagttcaagacca 112560 gcctaaacaa catagtgaga cccctgtctc tacaaaaata aaaaataaaaaactatctgg 112620 gtgtggtggt gtgtgcctgt agtcccagct acttgggatg ctgaggtgggaggatcattt 112680 gagcacagga agtcaagacc acagtgagca gtgatcaggc cacttcactccaacctggat 112740 gacacagtga gaccatgtct ccaagggggg gaaaaatata tacacacacacacacacaca 112800 cacacacaca cacatatata tgtgtgcatg gtgtaatata tgtatatatataatcattac 112860 tcttaatata taatatagca tatattttaa tatatcatgt attttacatattattaattt 112920 ttaaagctta aatactaaac tatctggttg ttctgcctta tgatgtttagtcatttgtga 112980 gccctaaagt gatggagtct ttaagtacag acatacagga cagcaaataaaaccagacta 113040 aatgtagcct tggatttcat attgaaattt tttaaaaaat aaatctagtgtttggaaaga 113100 tttatgattg aagattttct ccaacttgtt gaatacagat tggtggtcaatataaaaagg 113160 atatgaatgc tcagttttag aaagtacatg gtactaagaa gataaaaatgggtgtaaggc 113220 caggtgtggt ggctcacggc tgtaatccca gcactttggg aggccacggtgggtggatca 113280 cctgaggtca ggagtttgag accagcctga ccaacatggt gaaaccctgtcactactaaa 113340 aatacaaaaa aaattagctg ggtgtggtgg tgggtgcctg taatcccagctacttgggag 113400 gctgaggcag gagaatcgct tgaacctggg aggcggaggt tgcagtgagccgagattgcg 113460 ccattgcacc actccagcct gggcgacagg agtgaaaatc tatcaaaaaaaaaaaaaaaa 113520 aaaaggatgt aatactcttt ctagaatagt ttgactttta gtttcatatacttgtaattg 113580 gcctctcatc tgtctatttc tttggaacat caaagttgag aagatttgtagaagatgtta 113640 ataaatctgt ggtaatgaga gtagaaattg tgttgaagtt taaaggaaagaacaaacatg 113700 ataagatatt ggtagtacat tattagaata tttctgttta aattaaaatagtatttcagt 113760 tattataaca tttgaaaatt cggttggaga atgttttaaa gttattaagactgccatctg 113820 ctagtaaaaa ttattgtttc aaacttcctg aggcctccag cttgggtgacagagcaagac 113880 tctgtctcta aaaaaatgaa acaaacatct tgaggctgga aatatttgaatgaaaaataa 113940 tttttagttg taaaatggca atgaagtata atgaattttt ttgtcaattgagatgttaaa 114000 acacttttat taataaaaca tgaaaaaatt gttagagttg accatacttgcttcttgctg 114060 ttcggcacaa attctgttct tctgactttt ggaaattttc ttttactgcacttatttttg 114120 aatcttcatt ttctctaaat gtgtctttct gtaaccttgg ttgtcttacagttttagttt 114180 ttaatagtaa aatgaaactc tgagaatttc atacacgtgg aacctgcttttcctggaaca 114240 atcacagcca caacttttat caatatatgt ggtttggaac tgaaaaacttatgaagtgtc 114300 ttttattaac tctgcaattt tttaaacctt tcaagagatg aaacgaataaagaagaagat 114360 gaagatgatg aagaagcaga agaggaggag gaggaggaag aagaagaagaggatgaagat 114420 gatgatgaca acaatgagga agaggagttt gagtgctatc caccaggcatgaaagtccaa 114480 gtgcggtatg gacgagggaa aaatcaaaaa atgtatgaag ctagtattaaagattctgat 114540 gtcgaaggtg gagaggtcct ttacttggtg cattactgcg gatggaatgtgaggtaactt 114600 gagttttagc tagtgattat tacctaaaaa caattataaa atacttttctatttaaaaac 114660 ttaagtaatg gattacagat ttattagtat tctaattgag gtcatagtaccttataaaaa 114720 atagttttta gcatgtaaat acctcactta gtgacttaca attaatgttttcactactta 114780 gaagatatcc ctaatgtcta gaaaaccgag gtctacacac ttgatttttatgtactacat 114840 gtagatcagc agacaaaaca ttgttgaaat gatcagaact tctggacatctacaaaccaa 114900 ttatcaggct gcttgctgtc ttgcatttta aactacattg ccacagtaacctcttttttg 114960 tattctgaag tcatttcttt ccttctgtag tcagggattt aaaatcctaggaaacattag 115020 ctattttaag aaagccttat ctccttatta acagctattg cattacagccttattcagaa 115080 gtctcagaaa tttattagta tctttgaaaa aatctggttg aacatgtaagcagacctgct 115140 tcactaaaat agaatgcact ttttgactgc tcctaaccta gggaactgaaaggcaagagt 115200 gaaatttggt tttaagtcat gttcaatgac ttaagcattt ttcttagataaattttcttt 115260 ggctttccaa agaaattttg tattttgatt ttatccaaaa ttggcaagccagttttggga 115320 tctattcatt cagttatctt ttaagaatca tttattttag aatatttgatagtatatttg 115380 aaatgtagcc tataagacag atacctggaa agattaacca tagaagtttaagttgtggtt 115440 tcatggtgta cctagtgtct tcaaggagga atacagttct catgctttatacttaaatta 115500 tattctgtgg tttgggaagt taataggaag gcatcatctc aaattctgtgtttgagtgtt 115560 gcaaatgaaa ttttgattct ctttaatcac ttcccacaat gccctttctctaaagaaaat 115620 cacttctgag agttttgagg ctgtccttac taacttttca ttctactttcacatacatat 115680 aaatgtactc atagaaaata tatagtattc ttacatgcat gtgttttaaataaattatac 115740 tatacaaaat tctgtttctt aaattttttg ttgtcctgtt gtgtaataggaagctcatga 115800 tctaatctga ttacctttct tattttgaga aaagtaccag tccgatccattctgatgctt 115860 ggaagaaaat agcaaatagc aaatagcaac ttaagtcatg aggagatgttcagaagtttt 115920 gttttggtct cattctttat tatgtctaat caggatacag ttttagattttaagaatacc 115980 tatcgtcaga gcttagatgt gatagtcatc cagaaacata agtttttccagataccactt 116040 taacacactg cacaatttat cctttcacct gttggttctt tttttctgttttttgttttt 116100 gtttttgtct ttttttggag atagagtcta gctctgttgc ccaggctggagtgtagtggc 116160 acgatctcta ctcactgcaa cttccacctc ccaggctcaa gcgattcttgtgctgcagcc 116220 tcctgagtag ctaggattat aggcgcccac cattgtgcct ggctaattttcgtattttta 116280 gtagagatgg ggttttgcca tgtttgtctt gaactcctga cctcaggtgatctgccttgg 116340 cctcccaaag tgctgggatt acgggtgtca gctgttggtt cttatgctgcctttctggta 116400 tgtgattttc ctcacctgaa ggtctggcta ttaatgaata tattccaaagaaggtacttg 116460 agatctcaga tatagctacc aaaagaatat tttgcccaat tttttacttctagtcatttt 116520 gatcttacta aattatctta ccgaatataa atgtagaggg agttctacttctgcctagga 116580 tataaatgta aatgtagaat atgttagttt aaaattagga agaaaaaagctgggtgtggt 116640 gttgcatgcc tgtagtccca gctactgagg tgggaggatc acttgaacccaggaattcga 116700 ggctgcagtg aactatgatc atgccactgt actccagcct gagtgccagaatgagacctc 116760 atctcttaaa ttaagaaata cttggattag caaaaacgaa taattactgcataatgccca 116820 tgagatagag tcacaacacc ttttgcctct ctttttagtt tttctgattattttgctagt 116880 ttacatgtag aggcagtttt tcctcttggc ctttatgaag tatttgaaattctatgaaaa 116940 agggatagaa tttaggtgtt ctccaacttt cactgtatat aatcagggctcatttccttc 117000 ccttcttcct tccctccttc cctccttcct tccacggagt ttcactcttaatgcccagcc 117060 tggagtgcag tggctggatc ttggctcacc gcagcctccg cctcccgggttcaagcgctt 117120 ctcctgcctc agtctcctga gtagctgaga taggcacgca ccaccacgcccagctaattt 117180 ttgtattttt agtagagatg gggtttctcc atgtttgtta ggctggtctcaaactcctga 117240 cctcaggtga tccacctgcc tcagcctccc aaagtgctgg gactacaggcatgagccacc 117300 gtgcccggcc aaggcttatt ttctattaat tggctcttta aacaaaaaaggcttagtaga 117360 tataagaact gagtcattca tgaaactgtt ttacaattaa gatatagctttgttttgtta 117420 agaaaagatt gactaaaaga aagagaattc caggagtatt ttgattttagatggtagaaa 117480 gaatctctag taatttagtt tttaaatagc aggaagggaa aggaaagaatttaaattgag 117540 gcattcaagt ttttaataat atagggagga aaatgcgacc tagttcaacaagacatgctt 117600 gtgttactgt gaacgttgcc atctatttct gagaactcaa gtgcttgtatgctatgaact 117660 ttcaaaagaa gaaaacttaa catcttgatg aagacctgta ggatggatctctatacactc 117720 caacatcatg gagaaaacaa atgtacttca gtagattaag cagttaatcttcttagagtt 117780 aacataacta ttttatttct ttctgcaata atcagtctgt cttattgataaataagcaat 117840 aatttttatt tttgcggctc ctccagttct gaagctaatt taataaggactgcaacaaat 117900 cctcatcagc tgtatgacaa tttttctttt atttttaggg tcttcagcttgatctctaaa 117960 tttatactta acagatgtga aattggaaat gaagatttat gcgtggaggttcatgaaaat 118020 agttttatct tcagtggtag tcattcatga caagcagttt gggagatttgtgtggaccta 118080 tttccatggt ttttttaatc tgaaacaagt atttcagatt aaaaaaaaagagagagacag 118140 acaggacctt gttctgtcac ccaggctgga gtgcagtggt gcaatcatggctctccagcc 118200 tcgacttctt gggctcgagt gatcctccca cctcagcctc tggaataggtgggactctag 118260 gtgtgcatca ccacacctgg ctaatttttt atcttttgta gagatggggtctcactatgt 118320 tgcccaagct ggtttcaaac tcctgggctc aagcagtcct cctgccttggcttccaaaag 118380 tgctgggatt atagacatga accaatgctc ctggccatga aacatgtttttccggtaaag 118440 agattttctc aaataatgta agagtgtctg tctttttgtt taaaaatactgtttttaggc 118500 caggcacggg tagctcatgc ccataatccc agcactttgg gaggccgaggcgggcagatc 118560 acctgaggtc aggagttcga aaccagcctg gccaacatgg cgaaacctcatctctactaa 118620 aaatacaaaa attagccaga catggtggca ggtgcctgta atccgatctacttgggaggc 118680 tgaggcatga gaatgacttg atccaggggc acagaggttg cagtgaaccaagatcacacc 118740 attgcactcc agcctgggtg acagagcaag actccatctc aaaaaaaaaaaaacaaaaca 118800 aaaaacaaaa aaatcaattt tcttgttaat ttttacattt ttcttttttttctaactaaa 118860 atgctgtttg tttgtttgtt gtttgttttt gagatgaggt tttgctcttgttgtctaggc 118920 tggagtgcag cgccgtgata tcatctcact gtcacctccg cctcccaggttcaaaagatt 118980 cccctgtctc agcctcccaa gtagctggga ttgtaggcac atgccaccatgcccagcaaa 119040 tttttgtatt tttagtagag atggggtttc accatgttga ccagactggtctcaaacgcc 119100 tgacctcagg tgatccacct ctgcccccgt aagtggtggg attataggcgtgagccactg 119160 cgcccgacct gtataaatct ttaatataat tttcatgtat gtccctatttcacagctgaa 119220 aatgttgatt tttgttatac aaaggttaac atttgggcaa gtttgttttctactgttgtg 119280 atgaaaaaaa gtcatatttt aacatgttca caccacattt actggaaagggcttgaactc 119340 tttaaccaca aatattataa tactcttaca gaatttttct gatgcagtgacagtatctaa 119400 aactattgtc tagcactata ttacttacta tgtcaccaag cactaggcatgtttggagta 119460 tgctcataaa ttatttttta aactcttcta aaaataacaa tttagaaaaatacataaaag 119520 tagaaagata aaattattgc tggccggccg ggcacggtgg ctcacgcctgtaatcccagc 119580 actttgggag gccgaggtgg gcggatcacc tgaggttggg agttcgagaccagttcgaga 119640 ccagcctgac caacatgaag aaaccccatc tctactaaaa aaaaacacaaaattagccag 119700 gcatggaggc acatgcctat aatcccagct actaggtagg gtgaggcaggagaatcgctc 119760 gaacctggga gacggaggtt gcggtgagcc gagattgcgc cattgcactccagcctgggc 119820 aacaagagcg aaactccatc tcaaaaaaat agaaaaaaaa taataataaaattactaata 119880 ttatagtgtg ttctctttta gtcttttaaa atgtctccct acatttaaaaaaaatttagt 119940 tctgcttaag tagtttttaa tagtaggctc tctatttaac ttttcttagtgatgttaata 120000 ttttatagag aatcttagca taattatcta attgatccct ttcactctcaagtttctaag 120060 tcgtgtcgta aatatgggat tgatttttga aagtaagggt acacattgaaattaacatgc 120120 cgtaaactag ttgagtgtgt tggatttctt tgtttagtaa caactgagtgaaactatcta 120180 gactaattgg catggaattt aaaatacatc aatattttgt aatagtgtatcaatactgat 120240 tgaattttct cgctcatttc caacttggct ttctgtaatc aaagagttgcaaatttagtt 120300 agagaataat tttttgtatg ttgaagatca ccagctgcat taatgggaacccaaaagtaa 120360 aagagacact gtaaaacttc agttgtaaaa ctcttaaata agccacaacagttctagtgc 120420 atgttaacag ttacagagaa ctgaaaataa gctaaattat gctaaatatattatttgaaa 120480 ttaaaaatgt gttacattta aaaaatattt tcgccaaaca aattaaatcctcttaggagg 120540 gaatgagtta ggggatgaaa aaacctggta aaaggctttc acaaatgagtaggctcaatc 120600 ctattttttg ccatcattca gttgcttttt atcacacttt attcctttattttccaaaca 120660 taagttgaat acccactatg tgccaaatac tgtgcttgtc accaaagataccaagaagtg 120720 gtccttatta aggaattcat aataataata aattctttat taaggtattgatcctaccac 120780 cccttacctc ttcaccccag ttaatgcact cagtcttccc ttgatattgcttacattccc 120840 tggcattatt attataatca cttcttttga ctcaagctgt tctttttctctccttttgct 120900 ttatagtact catctgacaa aaccacaaga accttgatta aatctaaccctccagctcct 120960 tgcctgcacc tgtgcagccg atcgtggttg gagtaaagca agcaaccacaatgactgttc 121020 ttattttaaa ttcattactg tgaacctcaa gtgggtcctt aattctctatgtccttcttc 121080 cattcacttt cctactttct tagaagacta tttcatactg ctgcttatgtcttcacatcc 121140 ccaacatctc ttccctatcc tcattttcag ctggtgacct tgctttgtacctactgagag 121200 atagaaacaa gaaaaacatt tctacacact tcataagatc tgcctacctaactgcgtttg 121260 ctgccacatt ctctgtgttt tcatctttag ccatagatat acagtcatgctactgtctga 121320 agccagtctt cataagcatc ccttcttgac tgatcaaaga catggcccattttcttgctc 121380 tgttctgagt cattcccact acttacagat ctgtggttat ttgtcacatctcaaggaaaa 121440 tcttttctgg agcctgactt ctactgtgac atttcttcgc tccattttcattcagcagaa 121500 ctgtttgaaa gttacctaca cttgttactc agtttctctc ctttttctgtgtaaatccac 121560 tctcatcagc cttttgttca ctgtgtctcc acaactgtgc ttaccaggaacactagtgac 121620 cctttgtaga taaattcctc ctgcttttct tgccctctta cgtaccttatcagtggcttt 121680 acagtcagtt gatcactcct tccgtgatat gctttgcttg ggctccagctgctccagcta 121740 caaccatttt tcttgtttat tttccttttc ttcctttctc aaaattaagaaaccactcct 121800 caattatctt ttctgtcttc ctcatcttta tgtgttttct cccttctaatttttccccta 121860 tttctttctg atatccattt aacatttttt tggtttctgt tcctatttttccaatcctaa 121920 cttgagtcac aattctgata ttgaattttt ctaaattatt tttccgagccaaaggaattt 121980 gatctggaat ataactttca cctaggactc tgggtaaatt ctttcccttcaactctcctc 122040 cccctccttt tctttttttc ttcatcgttc aagctttcta cattaggactacgttatgta 122100 ttattacaaa agtgaactta attttaaaat atgaaaaaaa tttctgtttcatatctattc 122160 tcctaatatc ccttaccaca tctagaaaag tattcatttg gcatatgataagtactaaat 122220 gaatgttgaa ctaaattgga cctcctgaca acaaaatgga agtagagcagaaggtaaaat 122280 ccaaataaaa gccactggtt ttttaaagtg atacctataa taggggattgggacagatat 122340 ggcttgaccc tctcactgga gctgcttctc acctcattta tatataactctgtgtttcac 122400 attctaagga actgtcaaac ttttccacag agacgacacc gttttacattctcaccagca 122460 atgtgttagg gttccaggtt ctctgcagcc ttgtcaacac ttgttattctctgtttatct 122520 cattgtgctt ttttgatttg cattttccta atgcaacatt ttttcatgtacttgaaatgt 122580 agagttgaac attttttcat gtacttatta gccatttgta tatcttctttagaggaacgt 122640 cccttcaagt ttttgccatt tttgaattgg tcagttgttt tgttgttgttgaattatagt 122700 tcttcatata ttctagatgt tattccctta tcagatacat gattgcaaatgtttcttttg 122760 ttgcctctgc ttttgatgtt atttccaaag aaacattgcc aaatccagtgttctgaaaat 122820 tttcctttat gttttcttct aagaacttta tagttttagc tgtaatgtttacattgttgt 122880 tctaatctga gttcatattt gtatatgata tatgtgacct ctttgggttacataaagagc 122940 tcatctctta gcaaacaaaa tggagtcgag actgagagac tggaatgaatttgattggaa 123000 atattaattt tctgttactt tttagctatt cttagtctcc ttttgtccttaaaaaatttt 123060 taagtcactt ttcagcttaa tttctttgtt ctttcctgct ctaaaatgttttgtgtcctt 123120 catttttttt ctttcttatt tccatgtaat ctgatccctg ctgtacttgagttgtgtatg 123180 ccattggatt taggaatatt gaaatagagt acaaagttgt tctactcattttctttgaaa 123240 taattgtcct ggtaatgata gaaagcaagg taccctggga agttgtctgcgttgtttcta 123300 tagtacagaa gtcagaggcc tataaaatga ccatttgaca gtggtttccattatctgtaa 123360 gtttgtatta aaagagagtg gcttttgagt taaccataga ttttgagtacaaatgaacat 123420 aaacatttcc ttcatgaaaa aaagatgata attcagttat gtgttgtactcaaatgggct 123480 ttaatttttc tttgtgtatt catcttagaa tgtttactaa acttggacctgtttcattca 123540 gtagtttcgt agattggcag gtacattgat atttttgtct aagtacgcacaaataactaa 123600 ttccatacat ttatcaaata ttaacaaagt acacttttgg ggaatttaaaaaacttagca 123660 tctgcattac aatatgcaat taatggcaca gttaaccatt tgcagttgtctttgtacgct 123720 ttggtaaaat gctgtattag tgttcttatt attacatatt acctctgcctaaagacctat 123780 acttccgttt cattttatag cgttacattg cttaggaaaa cctattgatataattactat 123840 accattccgt tgagctgttt atgtcaactc ttaaatatga ctatgctataatatttatgg 123900 ttctgtcatt ctgctgttat attcttaagt gtgaggaata ccatcgctgtctctctgatg 123960 tagatttaag tgttatatga agtagaaata tgaaatgttt atatcatacttaggaatttc 124020 attttatttt attttatttt gaagcggagt ttcgctccgt cacccaggctagagtgcagt 124080 ggcatgatct tggctcactg caacctccgc cttctgggtt caagcaattctcctgcctca 124140 gcctcccgag tagctgggac tacaggcgtg tgccaccatg cccggctaatttttgtattt 124200 ttaggagaga tgaggtttca ccatgctggc caggctggtc ttgaaatcctgacgtcatga 124260 tccgcctgcc ttggcctccc aaaatgttag tcttacagat gtgagccactgcacccggca 124320 ggaattttat ttttgaataa tatactactt ttttgtttca ttttattgtagatacatgat 124380 taaaagtatt aattgtgatc agttgttatt ttcatatatc ttgttttagatagtcagatt 124440 aaaatacata taaattgaga ttatataaaa ttcataaagt aggtaaatattgataaaatt 124500 gcttatgaag tttataatta ataatgatta acactgcttt ttatcttgacttggtgtttt 124560 tttctattat tttgtcatac ttttatttat ttaattcagg tggggaaatccctttccata 124620 ctaagacagt acaaatttcc actttagatg tgaggttctt aaaaactaagtaagttaatt 124680 atatgacata cattaacttt tttgattata gagaggcaag atataaaatacatattgtgc 124740 aacaattatt aaatgaatct attcattata gaaagcaaaa tatagatgattttaacattt 124800 acttaatggt tggttattag atttttatct tatctataca tatttcaaagagtgaggttt 124860 caagtttata ttaatttgat tttacttacg aagttatttt taaaatacctttattctgca 124920 tgctgtttat ttttaaatct cttaagactg tgctttttag gtatatattaactatatgct 124980 gataatattt cttttaaaat tgggtttaag aagaaccaaa agctgaattgatgtgggcat 125040 attaaccatt ttggtagtat atctggtcga ctaatgttct tttccatttttattattaca 125100 aaatgatttc ttacataatt tgccaatgca tctgtacaaa agccataaagttttcttttt 125160 agaaatccaa tttttctgtg gtgagagcaa gtatgctgtg gtctctgaatggtatctttt 125220 agccttgtat gaatattttt aaacatttga aaaatcaaat atttgtgtggtggcttcttt 125280 cagaaagttt cacgtttgta ttcacaattt gtcattactg tttagtattgtcttcttttg 125340 ttgaattgta agctcctcgc aggcaagggc tgtgtttgtt gattcattgctgagtaccca 125400 gtgcctagca gactctgtta ttacataata gataccttag aggtgtttgtcaaatgaata 125460 cgtccacatg tgtacttatg gtcttggagt ctcatataat ttttcattctacggccaggt 125520 gtggtggctc acgcctgtaa tcccagcact tcaggaggct gaggcaggcaaatcacctaa 125580 ggtcaggagt tcaagacaag tctggccaac atggcaaaac ccagtctctactaaaaatac 125640 aaaaattagc tggacgtggt ggcacacacc tgtaatccca gctactcagggggctgaggc 125700 aggagaatca cttgaacctg agaggcggag gttgcagtga gctgagatcgcaccattgca 125760 ctccagccag ggcaacagag caagactccg tctcaaaaaa aaaaaaatttttttttcatt 125820 gtacttcatt ttatgtaggt cagggaacat actagatctt cagaagttaaatgagtttat 125880 catgcatatg ttattttatg ttttgttagt tgaatgttaa gtgatctcaatagaaatatt 125940 atcagacaag tcatatatat accttggaaa tattttcctt agtactttagataaatattc 126000 aataagatac ttgtttagat tccatctgta gaattaatta cattgactgtattgataact 126060 gaattgaaaa agtataggct aatcactgtc ttaattttct aactcaatatttgtttttaa 126120 tatttgtttc aatacttttt gtttatttca gtatttcttt ttaaatggaagcatttagta 126180 atgctgtttc ccttgtgccc tgcaaaatta atttatttcc tgttattatgaaaaggccaa 126240 ttatgatttg taatcataat tcctaaaggt taagaccagt taaagccaggatagataaac 126300 tatcctggat tgtatatata tgtatatatt gtgtatatat atgtatatattatatacaca 126360 tatatataaa ataacaaccc agtctacctg aattttcaca ttttcagaaaactgtattgc 126420 ttgaatctaa aattgcaaga caatttattt tattttattt tattttacatatttttttga 126480 gacagagtct tgctctgttt cccaggttgg agtgcagtag tgcagtcttggctcactgca 126540 acctctgcct tctgggatca agtgattctc ctgcctcagc ctcctgagtagctgggatta 126600 tagacatgcg ccaccgtgcc cagctgattt ttgtattttt agtagagatggggtttcgcc 126660 atgttagcca ggctggtctg gaagcatttt aaaaccaaaa ggtttgtatttaacttactt 126720 actaagatta ttgtattccg ccagatgcgg tggctcacac ctataatcccagcacttcgg 126780 gaggccaagg tgggcgaatc actagagccc aggagtttga gaccagcctgagaaacatag 126840 tgacaccctc tctctacaaa atataaaaaa attatccagg tgtggtggtgaatgcctgga 126900 gtcccaactg ttcaggagtc tgaggtggga caattgatac tgggaggtcaaggctgcagt 126960 gagccaggat ggcgctaccg cactccagtc tgagcaacag agactctgtatgaagaagaa 127020 aaaaaaaaaa agaagattct cgtattcctt aagaatgtac tttcggggatgaatagatat 127080 cttacttgac gtggtacccc aaggagaaat tgagaaatat tttatggccttctatttctt 127140 agctgcagtt tcactctttg ccacttcctt tccagttatt ttgttttactttatttttat 127200 ttattttatt ttttttggag acagagtctc gctgtgtcac ccaggctggagcacagtggt 127260 gcgatcttgg ctcactgcca cctctgcctc ctgggttcaa gtgattctcctaccccagcc 127320 tcccaagtat ctgggactac aggtgcccgc cgccacagcc agctaattttttttgtattt 127380 ttattagaga cggggtttca ctatgttggc caggctggtg ttgaacacctgatcttgtga 127440 tccgccctcc tcggcctccc aaagtgctag gattacaggc ataagccaccgggcctggcc 127500 tattttaatt ttttatgaaa ttcaagatat gtgaacttga gctaaaatttcccattttct 127560 atctgcattg actattttct tatctattat gtctaaggta aatttggtgttgtctctttt 127620 taatatatac taaatgtact gtatttttaa aagtctaaat ttataaagaagaagtaggat 127680 taactgtagc tttttgttgt agttctgaag taaattcttc tttgaagcctgtgcttcttt 127740 ggtaaggaat gagaaatctt gacccactaa tattgctttc ttaaattaattggcaccagt 127800 aattatgcca gtctaagacg ttctttctta tataatttta atgtctgtaggaaatatggg 127860 gttttcatgt agagtttttc cccgcatgaa ttttattttt ttgagatgagtcttgctctg 127920 tcacccaggc tagggtaccg tggcatgcga tctcggctca ctgcaacctccgcctctcgg 127980 gttcaagctc ttctcctgcc tcagcatccc aagtagctag gactacaggcgcccgctacc 128040 acgcctggct gatttttgta ttttcagtag agacacggtt tcaccatgttggccaggctg 128100 gtctcgaact cctgacctca tgatctgccc acctctgcct cggcctcccaaagtgctggg 128160 attacaggca tgagccactg cgcccggcct aatttttatc tttttgttagagacggagtt 128220 tcaccacgtt ggccaggctg gtctcgaact cctggcctcg ggtgatctgcctgtctgggc 128280 ctcccatagt tctgggatta caggtgtgag ccaccgtgcc cggcctccccacatgaattt 128340 gctttttttt ttttttttcc ctattctttg aaaatatcct gtagagaaaatagaaggaac 128400 acttgggtag tattgtaatg ggtaattaca tatacggaag gaacatttcttatttttaaa 128460 agcaatgatc agttatagtt atttaatctt ctgaccatct ttatttttcttgtaagatct 128520 atcagacaac taaagtgcta tttaacgtct cattaggttt tgaaatacttgtctgccaat 128580 atgtttgtgt cagtgatctc agcttgtatt tggtaaggac atttgtaattcgaaaaagct 128640 tccccaacca aaattctcac atgttatttt ctccttttct tcctcttcacgccactggtc 128700 tctcactctt cactaaactt actctgaata gggcctggaa tgccccctaccgttattttc 128760 gtgttagaat cttttcccgg gcagtccatt tccttctctt ctctaattctccacaggagt 128820 gctttttggc aagcactgtc tttcactttc attgttggaa tttcttaacttgtgaaataa 128880 aggccctgat aaatttttaa gatatgctct agttctaaaa gttattagttttgataacag 128940 taacaatatt gtgtaactgt gttttataca aagagcataa ttggcaaggtaaaaaaaaat 129000 gcaaaaactt agtgtcaaaa caagggcctg aattaaacaa ataaaagcactgagaaagta 129060 gaatagaaag aagccaaata acagaagagt cataatatta gctaatatttatagatctgt 129120 taagttagat ctataaatat aatgtaatat tattatacat tatgtttgctgttagattac 129180 attattactc tatagtaagt tttcagggaa tttcttaaaa ccatacagagtattccatga 129240 gaattttttt tgttttgttt tttgagacgg agtcttgttc tgtcacccaggctggagtgc 129300 aatggcgcga tctcggctca ctgcaacctc cgcctcccgg gttgaagcgattctccagcc 129360 tcagcctccc gagtagctag gattacaggc atgcgccatc atgcctggctaatttttgtg 129420 tttttgtaga gacagggttt caccatattg gccaggctgg tctcgaactcctgacctcag 129480 gtgatctgtc cacctcagcc ttccaaagtg ctgggattac aggcgttagccactgcgccc 129540 ggccccatga gaattttaaa acaaaaattt tccagaatag taaggccatactgttaatta 129600 caactatgta tgtagtttgt ttcatctaat ttaagaattt atttttgtgtgtgtgtcctg 129660 cctgctccaa cacacttttg acatctaaac tcaactactt aacatttttctgacatccag 129720 tcctcttgta ccatccttct tttataattg agtagaagag aacatgattaccatatagga 129780 tttacagagc ttaggcagtg ggccccagcc tgtaccatac caagcatccatcattttatt 129840 atttatgtgt atgttcttgc caccttgttc ttaagagaat ttaaacaacatacattatat 129900 ctgagtaata aaggaatctt cctttatcac agtggcctta cccatgaggcagtaactgtt 129960 tttccttgtt tgatgtgttt ggtgttgcat gtaaaatata caaatgtgcaatatcatgca 130020 tgtattctta aattgaaaac tactatttta ccctacttca aaatataattgataataaaa 130080 cccaggttga attgagtcag ttttttattg atagtaaaac atgtctggtataaaactagc 130140 tacagaacag atagcctttg gttaagaaag gtagaacatg attataactttgtttagctc 130200 acattaatgt tatgtgcagc catcatgaac tgacttcagt ttctctcataaaaaattaga 130260 aagcttgttt tgttttcttt taaaaacaaa gttgatatct tgacttcttctttccataac 130320 tcttctaata gagcaacttc taggagttca tactgaaatc tacagaggatggaggtgttt 130380 tcttttcgtt tcccaccatg cccccgattt tggaaggaag taaattgctttcctaagcat 130440 tatacaattt tcgatattaa taggaaaggt aaaagtactg ctgcaaatatatggatagat 130500 gctacagtgc caggtagaag taccaaaagg agtgtattta gtagaagaatacttacttta 130560 aagcttttca aagtacttac catatatttg tacttacacc agtacttaccatatatttct 130620 acaaaaaaac ccatatatgg tatgtaccat tattttttat tttgtttttaaattgtagtc 130680 ttattttata cagaaataaa ctaagcctta gattgagaat gagttgatcaggaccacata 130740 actaggaagc agggattatg ttaagtctgt cctaaagctc attccattccctcttattct 130800 tattacggcc ctcagcatct tccagcatat aaacagaacc ccttgcatggggatgaatcc 130860 tcagcttttc tggcttttac tggctacaag ccagtagttt gtaccagcagagtaactgcc 130920 caggagtttg ctacagatac tttccaggcc tatgctagag taagaatataatttattagt 130980 caagtaatga ctcaagtcat ttacttcttg atttggggac tttgttacttccttttaata 131040 cctctaacaa tgaggacatt ttaatttaaa agctcgtttt caggtgtccatgttctagag 131100 ttttgtgttt tctatgagta atgtatataa ctttgaacac aaaaagagaatgtcaattag 131160 tttttaatgt ttacttcttc tagctgttat aattcttgaa tgaatgaagaacgaataaac 131220 tgtcattctt ttttttttgc tttaaacaaa tagttttatt gcagttaatagtaatttgca 131280 ccctatgaaa ttgtgaagac catcagcatt gaatcaagag gccatcacagctgttgactc 131340 agctgtctgt acatgtaggg agcatttcag aatactgaaa aactttgcagagtaatgttt 131400 ttctggtatt ttatgagcaa gcattttttt ttttctagtt gattttgcattatccatacg 131460 ttacaatatg gtgcttttct tttcttattt cttttttttt tttttttttgtgaaacagag 131520 tctcgctctg tcgcccaggc tggagtgcag tggcgcgatc tcagctcactgcaactccgc 131580 tcctgggttc acacgattct tctgcctcag cctcctgagt agctggggctacaggccccc 131640 accaccacgt ctggctaatt ttttgtattt ttagtagaga tggggtttcaccgtgttagc 131700 gttgatggtc ttgatctcct gacttcgtga tctgcccgcc ttggtctcccaaaagtgcta 131760 ggattactgg tgtgagccac tgtgcctggc tgtaatacgg tgcttttctattacacaatg 131820 ccatttaaaa ctgaagctat taccttagct ctctgatgtg tctagagaaaaattgcctac 131880 ctgctgctta agaatgaaac aaagtaaaac aaaatgtaga aaatactgtgatttatctta 131940 atacttactg tacagattca gtttctcttt gttgtagccg agtaagcagtgctagttgag 132000 gaatcatgag atcatgctct ctagtcttgg ctcggccagt agctaactttaagtccatgg 132060 gcaagttatt acctccctct ctttgtgcct tggtagcttg ctcttcaaaccaggaagctg 132120 agatgatttc caaggtcctt tcccttctaa aatgtcaaga ttctatatgagtttctcttt 132180 aatatggcat aggttatttc cgtggtatat attgtacata tttatattaatttcctttaa 132240 ttttagatac gatgaatgga ttaaagcaga taaaatagta agacctgctgataaaaatgt 132300 gccaaagata aaacatcgga agaaaataaa ggtaagtgct tgtttttactacacactatt 132360 agctcttaaa aagaagtttt cctccaaatg atgcaattaa atatgtgttagtgttacact 132420 ttattgaaat agcacatttt tcaaatgcta gatttgtata taattaattttgtcaggtta 132480 gggaaagcat tttagaaaca gtactggatg atctttgaga ctttttcagtcccaacatta 132540 tataaagcca ttcaaccaga ttttatttat ctatttctgc catttccttcccctctagtt 132600 cccaaccagg agccataagg cagtgtgact tggagagaca taggttattattgtagctgt 132660 aaaccattcc aaggaagaaa aggctatctg gcaactcctt tttctcttcttctttatctg 132720 attatagtca gtggcttaga atatattcac aagatttttt tttttttttggagacagagt 132780 ctcgctctgt tgcccaggct ggagtacagt ggtgcaacct cggctcactgcaacctccac 132840 ctcccagttc aagcgattct catgcctcag cctcccgagt agctgggattacaggcacac 132900 accaccacac ctggctaatt tttgtatttt tagtagagat ggggtttcaccatgttggcc 132960 aggctggtct cgaacttctg acctcaggtg atccgcccgc cttggcctcccaaaatgccg 133020 ggattatagg tgtgagccac cacgcccagc cccacaggat cctattaaccacgaaaatgc 133080 catgaggatt tacttaggag ctcatatgga aaattaatgt tacttttatgtatgatagaa 133140 ttattcattc taagactaaa ttttcctaaa tgtttgatta gcatcttctatgcatctgga 133200 atgatactag atatgtacca tccttaggag aattactctc actcaaaatttatagtgctt 133260 actgctaact catgcttcag agagcttatt tttcctataa atcaacaaaatttttaagag 133320 tagtttccag gaaatatatc ggcctttgct tatctatttg taaacaaataatttgagaaa 133380 atttctcatc ttttctcaaa acattaatat atcactgagg taggaaggttgcccctgact 133440 tgactttaca aaagtacctg gaattttgtc attcccaacc cacatatagtgagattttaa 133500 aatgaccacg ttgcaatcgt ttcttttttt tcttgttttt ttgagacggagtttcactct 133560 tgttgcccag gctggagtgc aatggcgcga tcttggctca ccgcaacctccatctcccag 133620 gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggcatgcgccacc 133680 accccggcta cttttgtatt tttagtagag acggggtttc tccatgttggtcaggctggt 133740 cttgacctcc tgacctcagg tgatccacct gccgcggcct cccaaagtgctgggattaca 133800 ggtgtgagcc acctcacctg gcctcttttt ttagttagta tttaatttaaacgtattgtg 133860 attttaattc tgaagagcaa gttgtaggtt tgctagtcct aatcacctatctgattgcta 133920 atttgattcc cgttattaaa gtataaatac aaaacctttt gatcatcctgtgtagtattt 133980 tgttaagtag ctttaaaaat ctggctgttt tgaggggctt cttatgttctttctttttat 134040 gttaaatatt taacaacata ttctgtaata gtgagttaaa agtggacattaacattgttt 134100 ctgataaatg ccttatgata aattacgaca tacytttttt cttaacctagaataaattag 134160 acaaagaaaa agacaaagat gaaaaatact ctccaaaaaa ctgtaaacttcggcgcttgt 134220 ccaaaccacc atttcagaca aatccatctc ctgaaatggt atccaaactggatctcactg 134280 atgccaaaaa ctctgatact gctcatatta agtccataga aattacttcgatccttaatg 134340 gacttcaagg taaacataac aatcgttctg ttgtgcaagt atttgattttaatttatgag 134400 tcaagttcta taaaggtaat tcagtgacat taccaactac tgttttttctaccagagttt 134460 tgtttgctct cttattacag gttgaatatc tcttaactga aattcttggaaccagaaatg 134520 ttttggattt cagatttttt tttgattttg gaatatttgc attccacaagccaaatccga 134580 aaattctaaa tctgaaatgc tccagtgagc gttacctttc agaatcacgtcagcactcaa 134640 aaagtgtatg attttggagt cttcagattt tggatttggg atgttcaacctgtacttgaa 134700 ataaaatcag ccattgattg aggcctcaaa ttttttatta ctagtagatccaagggcacc 134760 ataaaatttt ggttaacatt taatattcaa cttttagcat tgttactttagttttttaat 134820 cttttctgtg ccatcctttc tgttgcaatg taatgcatag tttgtgttatacaacatcag 134880 accataaaag tatactgaca agttgttgta aagaacacca aactttgtttgtcctgatta 134940 tgactattat gatctctgtt tctttgagac agggtctcac tctgtagcctaggttggagt 135000 acagtggtgc aatctcagct cacttcagcc tcaacctccc agactctaaagatcctcaca 135060 cctcagcctc ccaaggagct ggagctgcag gcgcatgcca ccatgcccggctaatattta 135120 tattgtttct agagacgggg ttttgccctg ttgaccaggc tcgtctcgagctcctaggct 135180 caagcaattg gcctgtctta gcctcccgag tgctaggatt acaggcaggagccactgcac 135240 ctggactgac tgcttttttt ttttttttga gacggagtct cgctctgtcacccaggctgg 135300 agtgcagtgg cgcaatctca gctcacttgc aagctccacc tcccgggttcacactgttct 135360 cctgtctcag cttcctgagt agctgggact acaggcgccc gccactgcgcctggctaatt 135420 ttctgtattt ttagtacaga tggggtttca ccatggtctg gatctcctgaccttatgatc 135480 cgcccacctt ggcctcccaa agtgctggga ttacaggcgt gagccaccgcgcctggcctg 135540 acatgacctc ttttatgatg ggattattgc ttagtattta aggcaaaatgaagtataggt 135600 aataaaagca atgaggattg gagcagagat ttattaaatc tgactggcagggaaggggac 135660 atatctatga gataaaagaa tggatctcca taaaaatatc ttatcatttctcataactta 135720 aaagaggttt catttttact tattttgaat gttaatagaa gctagtttttgtttcttatt 135780 gtagatgtgt gtttatatta actcattatt ttataaacca aatgctgtactatattttta 135840 tttactcacc actgtactaa ttagggcaat atttttaaat tgatttttaaaaaattttcc 135900 catgtatcct acatgcaaat agtgcatgga aaatacatgt atagtttaaagaacagtaat 135960 aatatggata ctagtatgtc tccacagtag caatcaaata ctatttacttaaaacatgta 136020 gtatgtctcc tcccagaagt aattatagtc tggacttttg tgtaatccttgctttacttt 136080 tcttcagaat tgaattatct atatttatat tcctaagtaa tttttattttacttcgtagt 136140 atgtctcctc ccagaagtaa ttatagtctg gacttttgtg taatccttgctttacttttc 136200 ttcagaattg aattatctat atttatattc ctaagtaatt tttattttacttcatttttt 136260 tagggtgttt ttttgttttg tttttgtttt tgaggcagag tctcgctctgtcacccaggc 136320 tggagtgcag tggtgctatc acgactcaag tgacctcctg ggctcaagcgacctttccat 136380 ctcagcctcc taagtagctg gaactacagg tgtgtgccac catgccaggctattttttgt 136440 agtttttgta gagatggggt ttcaccctgt tgcccaggct tgtctcaaactcctggactc 136500 aagcaatcca atccatctgc ctcagccttc caaagtgtta ggattataggcatgagccac 136560 tgcacccagc ctgtttttta gttttttaaa aattgagtaa caagcataagcctagtttta 136620 taagaaggct cagatgaagg ctttctgaga atgttacctg aatagagattctaagtcata 136680 ctctgatttc agatttagct tctctgagag tttacaaaag cccctctagagaattcagtt 136740 ggacaggtca taagccagtt ctgataaacc taggggtcca ctacagttagaatatctgac 136800 acatttggct gggcatagta gctcactcct gtaatcccag cacttttggaggccaaggcg 136860 ggcagattac ctgaggtcag gagtttgaga ccagcctggc caacatggtgaaaccctgtc 136920 tctactaaaa aaaaaaatac aaaaactttg ccgggtgtgg tggtgggtgcctgtaatccc 136980 agctacttgg gaggctgagg caggagaatc gcttgaaccc aggaggcagaggttgcagtg 137040 agtcaagatc acaccattgc actctagcct gggtagcaac agcgaaactccatcttaaaa 137100 aaaataaaat aaaaagaata tgtgacacat tcgcagtgga tgttatgtgggagtgtgctc 137160 agtcgtagta gcagactact ctatatatat tctcattcta ggcttatggtgatttacccc 137220 tatttttcac aagtaggtca gaatgtttct gctggataaa atgatgtcattagctgatct 137280 aggtagtggc acataagaca tggaagggag agctggaatt tcattgacacatagttgaaa 137340 caagataaat acagatttta aaatccagtc tgggtcactc aggcattcaaagactacaga 137400 gaggcggtaa ggtagcttaa tgataatagc ctgcagtatg actttctttggaaataacac 137460 tgctgtagtc tggactggtt tccctctata tctgttgcac agctattttcctagggagat 137520 cccattctag ggatctcctt taatgtagtt cttggatttc tttgtttttggcatctcagg 137580 ttcctcccca cccccatact ttcattttgg tggagcacat tctctagtagcttctaaaga 137640 acaagtgcat gagatggaat ttttagagat tggctgcatt taaatatctgtatttatttt 137700 tgcaaatatc tttattctat tcatagtttg gctggattgg aagtttttccttcagtattt 137760 tattggcagt gttgaattgt ttttttgctt ccagtattgc ttttgtgaagtccagagctc 137820 ttccaattcc tgattctttg tatgtgactt gttttattct ttattttttgcttttctgtc 137880 tctggaatct tttttttttc ttttcttttt ttgagatgga atctcactctgttggccaag 137940 ctggagtgca gtggcacaat tttggcttac tgcagcctct gcctcctgggttcaagtgtt 138000 tctcctgcct cagcctcccg agtaggtggg attgcagggg cctgccaccacgcctggcta 138060 gtttttctat ttttagtaga gatggggttt tgccattttg gccaggctggtctcgaactg 138120 ctgacctcaa gtgatccact cacctcagcc tcccaaagtg ctgggattactggcatgagc 138180 caccatgccc tgctaggaat cttgaatctt tgtttttcgt atccagaaatttaactctga 138240 aatatatcta cacaaggcca aaggaagaga acagatatat ccacagtacacttggctgtt 138300 ctctttcttt agccttgtgt agatatattt tcatcccctg tgatgcctacttgctagact 138360 ctttcattct agcaacttct gtctttctgt tctaggcagt ttttttgaatgattttcttg 138420 atgatttcct ctcctgcatt ttactgttct ctccttttta atttttgttcattgttacta 138480 tttttgagat gaggtgttac tatattgccc aggctggtct caaacttctgagctcaagcg 138540 attttcctgt ctcagcctcc caaagtgcta ggattacagg catgagctactgtcccagcc 138600 ctgccaaatt tttttttttc tttaattgag actgtgtttc actatgttcagcccaggctg 138660 ttctcacact tctgggctca agcagtcctc ctgcctcagc ctcccaagtagttgggtagc 138720 tggggtatag gtgcatgaca ctgcacttgg ctgttatctt tctttggccagccattattt 138780 gatattatac cttctgtaat atactctaat atgctcacct tttaatttcttgctttttat 138840 taattttttt gtctttttat ttctttgaaa ttttatctgc tttgtcttttaatccttttg 138900 ttgacttttt gctgtaatgt ttaagttttc aatgattttt tttttttttttttttttttg 138960 agactgagtc tcactctgtt gcctaggctg gagtgcagtg gcatgatctcggctcactgc 139020 aatctctgcc ctccaagttc aagcgattct cctgcctcag cctcccgagtagctggtatt 139080 acaggcgcct gccaccgcac ccagctaatt ttttgtattt ttagtagagatggggtttca 139140 ccatcttggc caggctggtc ttgaactcct gaccttgtga tccacctgcctcagcctccc 139200 acagtgctgg gattataggc gtgagccact gcgcccagcc tttcaatgaatatttttaaa 139260 ttaaactagt ttaatttttc aagagctctg ttttctgtgt gttttgtaccactctgttct 139320 tatagatgga tgcaatacct tacctttcgg attttgatct ttcaaaggattttataattt 139380 ttggtttttt cagttacctc taatttgctt ttttctgtgt tgttgttttgagctcttccc 139440 tattaagaat tttttttcct ggctgtctta tgattatgaa ggaaagactaaactgatttg 139500 gaaattgcaa gcatatggtt ggcacttgtt gaccttgagt ttcactatcgggtgatctgg 139560 ttggccatgt cctagggaat tcataatatg aagtctttag gtctttttctcttagactag 139620 ttggcttcca gagaaaagag ttccaatctc ttgactggag ggtggtaagagaatggtttc 139680 cagtgttcta ggatctagtt gaagaacaga gagtggggtg gaggattcttagtggttaga 139740 tatgttcatg aatcccccta atttcagcat cgcatctgta cctgtgccttcaacagttgt 139800 tagcatctcc caggctagga gccctctcct actgtcttca gagaataaagctctagaagg 139860 gcagtcacct tccaacctga agtgaggtgg ggatctaggc atctaagtaatttttagtct 139920 tcacccagtg ctccttgtat gaggctcccc actgcccttt ttaattttcataggcactag 139980 ggcagtcagt aattattgag gtttctatgg taaactgggt tggtttttggctttcctcac 140040 tgctggtatg aggtttagcc ttctttggtg cccgtcattc atttatctgctttctgactt 140100 gcaaattgtg ttgcttttct ttcgtatttt cccccatttt tataggtttatattatttca 140160 gaaattgcat ttatatacca tatatatgac atatgagtat atatacacacacacatacat 140220 ataacacata tacaatgacc ccaaaccccc ccacacacat atttatcctatatgtgtaat 140280 agacactcac atgtgtgcag tctttttaca gatgggatca ttaacatattacaaactctt 140340 tttttcccct acttagtggt ataacttata aatctttcca aatcggcacatccacatcta 140400 ctgaatagat atatcataat ttatttaatt agttgctaaa aaaagacattttgatccttt 140460 cccatatttt tcctgttagt tcaacaatga gtatcatatt tgcatacttttaacatttta 140520 agtattctct tcatattttc ttttttcagt gaattttatt ttttatctttggccacagat 140580 caaattgaag acatttcaaa tatttccagt taattaatga tcatcatatgaatgcatatg 140640 tgtatgtata tgtatacata aaaatttgat gccccttgtg agtagagaacacagacagct 140700 aagattctat cctcttgtcc tgagatgggt tgggaataaa actagaggtacagtggtagt 140760 tggagttctg tggagaaatt gaatgtctct ggagaataat cttctacattctggcagtgg 140820 aaatccccta gagtaaacta gatccgggat gaatcacgta aagcctatcagttaataaat 140880 cctactaaca gtagtgtcaa tcaagtttta atagggcatt cttaatcatctcttgttaat 140940 ctcagagaac ctacagtatc tataaaacaa gaataagctg ggcacggtggctcacacctg 141000 taattccagc attttgggag gccaaggtag gcggatcacc tgaggtcgggagttcaagac 141060 cagcctgacc aacagggaga aacctcgtct ctactgaaaa tagaaaacagttagccgggt 141120 gtggtggcac atgcctgtag ttccagctgc ttgggaggct gaggcaggagaatcacttga 141180 acccacgagg cggaggttgc ggtgagctga gatcacgcca ttgcgctccagcctgggtaa 141240 caggagtgaa actccgtcta caaaaaaaaa aaaaaaaaaa agaataagatgcagtgaaaa 141300 agaaaagaat aagatgcagt gaaaaagaaa agaacaagaa agacttggagataaaaatct 141360 aaaggccagg cacagtggct catgcctgta atcccagcac tttgggaggccaaggtgggc 141420 agatcacttg aggtcaggag ttcgagacca gcctaaccaa catggtaaaaccgtgtctct 141480 aataaaaata ttaaaattag ccgaatgtgg tggttggtgc ctataatcccagctgctcag 141540 gaggcagaag catgagaatt gcttgaacct gcaccactgc actccagtctgggcaacaga 141600 gagagaccct gtctcaaaaa aaacaaaaac aaaaaatcta aattaaaattcagtagagct 141660 gggcacagtg gctcacacct gtaatcccag cactttggga ggccgaggcaggtggatcat 141720 ctgaggtcag gagtttgaga ccagcctgac caatagggtg aaaccccatctctgctaaaa 141780 atacaaaaat tagcttggcg tgtgcctgta gtcccagcta ttggggaggccgagacagga 141840 gaattgcctg aacccgggag gtggaggttg tagtgaactg agatcttgccagcgcactcc 141900 agcctgggca acagagcgag actctgtctc ttaaaaaaaa aaaaaaataagcaaattcag 141960 tagagcatta agaattttaa atggaggaaa tcttcagaaa gtttaaaagagcagtgatgg 142020 aaattttaga gaaaagatag gttacataat gtattaatct aggaagggggaactatgaaa 142080 acagaggaga gaggccaggc gcggtggctc acgcctataa tccctgcactttgggaggct 142140 gaggtgggcg gatcacgagg tcaggaaatc aagaccatcc tggctaacacggtgaaaccc 142200 tgtctctact aaagtacaaa aattagctgg gtgtggtggc atgcacctgtagtcccagct 142260 actcaggagg ctgaggcagg ggaatcactt gaacctggga ggtggaggttgcagtgaact 142320 gagattgcac cactgcactc caacctgatg acagagcaag actcagtctcaaaaaaaaaa 142380 aaagcaaata actaaagaaa aattccagaa ctgacagaca caaatctttacattgaaatt 142440 gctcactagg taccgaataa attaatagag tcataccgta gtacatcattatgaaattat 142500 aaaatatcaa gaatgaaaaa atcaccacaa aggaatgaga aaccaaaggaaggttatggc 142560 ttataacccc ttaagcaaag aagcaataca ttcggaagga ttcagaactctgaaggaaac 142620 aatttttaac ctagaattct gtacctcgtg aaactctcaa tcaatgaagtttgagggtaa 142680 ggatgtattc agataagtaa ggacgatgaa tatttatatc ccatgtacctttttaaggaa 142740 actactcgat gagctctagt acagatgaga taaccaagga agaagaagatgtgagattca 142800 gttaacagtg gacctagctc aaaagaaagc agtgaggagg attccccggatgacagctgt 142860 tagagcagac ccagagagat accacccaga tgctactgca gaaaatagctctctagggtg 142920 cagagatagg atgaataagg ggaaattgga tacaacgctt aatttaatgaaataaaataa 142980 tgtaaaagag atacaaagga agatgtaaca tgcagaaaag tagttggaaactcttggaaa 143040 aataaaatgc tgtataagaa aggaagttta tcaaatgtac tacttatttctgcagggaac 143100 cacatttaca tatgttataa atactatatt gtaaaaatga aagataactatatatagaaa 143160 ggatggtaga ggggagatat gggtgttgat aagtaagaat ccccattgctcatagaggat 143220 aaactttata aataagtcag caggccaggg acgttggctc atgcctataatcccagtgct 143280 ttgggaggct gagatgagca gatcacttgg ggtcaagagt tcgagatcagcctgtctaaa 143340 atgatgaaac ttcatctcta ctaaaaatac aaaaattagc tgggcatggtggcatgtgcc 143400 tgtagtccca gctgcttggg aggccgaggc aggagaattg cttgaatccaggaagaggag 143460 gttgcagtga actgagatca caccactgta ctccagcctg ggtgacagagtgagattccg 143520 tcacacacac aaaaaaataa ataaaacaaa tagtatatta tgaatagtaagagatgacta 143580 taactatcag atgttaacac tttggaggtg gaaaaacaga tttttatcctctttttggtt 143640 tttcattatg agtctagatt tgtttttaat gatgtatatg tattgttttgataatttaaa 143700 aaatgtggcc atgcagacac acattgtaaa ggtgaaatgg tttcacaatcttagttatgt 143760 cagttatgtt ttacatttaa tgaatttagc taaagaacat gcatggatttctcagtagag 143820 agctatagtt aaggttgtaa gttctggagt taaatgactt agatttaactaccttggggg 143880 gtagattaac ctctaaaccc cagtcccttt ttttttgctt tgttttgttgtgttgctttt 143940 tgtttgtttg tttttcttat ttatttaaga ccctagtctc ttgaactataaagtggggct 144000 gataacatgt atctcgtagc attgttctga ggattgaatg agatggtctgttcaaagtgt 144060 atcaaaagta accagcatgt agtaagttct caggaaatgt tatcttaaaataacaataaa 144120 atgatttacc agaatgaaca cactgaagca gtaagttcca taattaattttacataagtt 144180 atcagtaact aaaattaatt atatctattc tttaaaacat catggaaatcattgattaca 144240 aaattattgg atattcatta ttacattgaa aaatgaagct ggccaggtgtggtggctcat 144300 gcctttaatc tcagcacttt ggaaggctga ggtaggcagg ttgctagagctcaggagttc 144360 aagaccagcc ttggcaacat gacgagaccc tgtctctaca aaaaatacaaaaattcactg 144420 gatgtggcac acctgtagtc ccagctactt gggtggctga ggtgggaggatcatttgagc 144480 ccaggaggtt gaggctgcag tgagccatga tagtgcctgg ggaacagagtaagaccctgt 144540 ctcagaaaaa aatagagaga gagagagaaa taaagaaaga gaaaggaaagagaaaggaag 144600 ggaggcaggg agggaaagaa aataaaacaa aacaaaacaa aggaaagaaaaatgagaggc 144660 caggtgcagt agcttgctcc tgtgatccca gctactcagg aatctgaggtgggaggatct 144720 cttgagccta ggagtttgag gctgaagtgc actgtgattg tgccactgcactccagcttg 144780 gatgagagag tgagaccgtc tctgatgaaa agaaaaatga aggctatagtttcataagag 144840 tataaacttg gggccaggcg tggtggctca cgcctgtaat cccagcactttgggaggccg 144900 aggagggcgg gtcacgaggt caggagatca agaccatcct ggctaacacagtgaaacccc 144960 gtgtctacta aaaatacaaa aaattagccg ggcgtggtgg tgggcgcctgtagtcccagc 145020 tgctcaggag gctgaggcag gagaatggcg tgaacccagg aggcagagcttgctgtgagc 145080 cgacatcacg ccacttacac ttttagcttg gatgacaaag tgagactcaaaaaaaaaaaa 145140 agaagaatat taacttggag gtagcacaat ctgggattat atcctgactccactattttc 145200 tcagatatat tatgataaaa atattatact tatataaact tggagaagcttttttctttt 145260 ttatttcagc tttattaagg tacagtttac ataaaagtga tgacttttatgtttaaaatg 145320 tgatgggttt tgacaaaagt atacagtgtt acaaccatca ccgtgatcgtaatagaacat 145380 ttccatcatt ggaagagcct cttagcctct ttgaaaccca gtttctttgcatgttacata 145440 gagataaaag ccagcaacct cgggtggctg tatacacagg acctaatatatacagtccct 145500 ggcacataga agacattgct agtgttctta tcatactcat catttttttttctgttattt 145560 tctagcttct gaaagttctg ctgaagacag tgagcaggaa gatgagagaggtgctcaaga 145620 catggataat aatggcaaag aggaatctaa gattgatcat ttgaccaacaacagaaatga 145680 tcttatttca aaggaggaac agaacagttc atctttgcta gaagaaaacaaagttcatgc 145740 agatttggta atatccaaac cagtgtcaaa atctccagaa agattaaggaaagatataga 145800 agtattatcc gaagatactg attatgaaga agatgaagtc acaaaaaagagaaaggatgt 145860 caagaaggac acaacagata aatcttcaaa accacaaata aaacgtggtaaaagaaggta 145920 ttgcaataca gaagagtgtc taaaaactgg atcacctggc aaaaaggaagagaaggccaa 145980 gaacaaagaa tcactttgca tggaaaacag tagcaacagc tcttcagatgaagatgaaga 146040 agaaacaaaa gcaaagatga caccaactaa gaaatacaat ggtttggaggaaaaaagaaa 146100 atctctacgg acaactggtt tctattcagg attttcagaa gtggcagaaaaaaggattaa 146160 acttttaaat aactctgatg aaagacttca aaacagcagg gccaaagatcgaaaagatgt 146220 ctggtcaagt attcagggac agtggcctaa aaaaacgctg aaagagcttttttcagactc 146280 tgatactgag gctgcagctt ccccaccgca tcctgcccca gaggaggrggtggcagagga 146340 gtcamtgcag actgtggctg aagaggagag ttgttcaccc agtgtagaactagaaaaacc 146400 acctccagtc aatgtcgata gtaaacccat tgaagaaaaa acagtagaggtcaatgacag 146460 aaaagcagaa tttccaagta gtggcagtaa ttcagtgcta aatacccctcctactacacc 146520 tgaatcgcct tcatcagtca ctgtaacaga aggcagccgg cagcagtcttctgtaacagt 146580 atcagaacca ctggctccaa accaagaaga ggttcgaagt atcaagagtgaaactgatag 146640 cacaattgag gtggatagtg ttgctgggga gctccaagac ctccagtctgaagggaatag 146700 ctcgccagca ggttttgatg ccagtgtgag ctcaagcagt agtaatcagccagaaccaga 146760 acatcctgaa aaaggtgaga aggaaaatgt gtatgttgac ttattttagggtttcccctc 146820 ttaaagtttc aatgatttca cagtatctct tgttataacc tgaggcgattaagtgtcata 146880 tttgtgtgaa catggtaaaa atggaaattt taaaggtaat ttgaaaatgaatagtggaat 146940 gcatttaaaa gcttgagaag gctttaatgt gctttgcttg agccatccatggcattttat 147000 tgtggaccag aacacatgct agaaattgca cccaggccca aatccaaacctgtttgagaa 147060 ttcattatat gcagtggttg actatatggc atgagcagct taaatctatttctgtaacat 147120 tgtttttgca attgtaatgt gcagtttctc acgaacattt tgatttattgacagacccct 147180 ccacccttaa ccaaatactg tatgtagggg tttggagcaa ccaactgtccaggcactgct 147240 ttcctcagac aaccgtgtca aactgatttt caagcctgac taacttgtgtgagtttgtaa 147300 aggaatttga tgctttctta gatgcatagc ttccaaattg aaggaccaatgtgtacgtta 147360 ataacctaca gtaatacttt tttgattttc cgtgaaattg ttaaaagtggaaattcaaat 147420 cggtaccttc ccaaagtatt agtgcctttc gatggtgcca tagccatacttctgtcatca 147480 tttttcttat aaaccacttc attcaggtat ctttaaatca gtatactctaggctgacacc 147540 tggctttgga acacactttt ccatcgtaaa gacagagcac tggagtatgttttttatata 147600 ccgtaaaaga ttttagaatg ctagctttag gttttcagca aagcttaaagagatatggtc 147660 tagatcaaat tagttaattc tatgttttct caggaatctt gacttaaaacatttctgttt 147720 taaaataaat taaaaaaata cttgcaatta aattgaaatg ttctttgctttttttcacat 147780 ttacaagtat aactactatg attttatctg tgccatacta tctcatgcaactgaactatc 147840 caggcatgac taatcttcaa aatgaaagaa tcctttattt cagaatattaggctttcaac 147900 agtaagattt tactggccag gtgtggtcac tcacacctgt aatcccagcaatttgggagg 147960 tctgggaggt caaggcggtg gatcgcttga gtccaggagt tcaagaccagcctggacaac 148020 atggcaaaac cccatctcta ccaaaaatac aaacgtcagc cagctgtggtggcgcatgcc 148080 tgtagtccca gctaccttgg gggctgatga gggagaatca cttgatcccggaaaggctgc 148140 agtgagccaa gatcacgcca ctgcactcca gcctgggtga taaaacaagaccctgtctca 148200 aaaaaaaaaa aaaaaaaaaa agaaaaaaca agaaaagaaa gaaaaaaacaggttttattg 148260 ttataggttt tcttgggggt tttttttttg agatggagtc tcgctctgttgcccaggctg 148320 gagtgcagtg gtgtgatctg ggctcactgc aacctccgtc tcctgggttcaagtgattcc 148380 cctgcctcag cctgctgagt agctgggact acaggcgtgt gccaccatgcccagctaatt 148440 ttttgtattt ttagtagaga tggcatttcg ccatgttagc caggatggtctctatctctt 148500 gacctcatga tctgcctgcc ttggcctccc aaagtgctag gattacaggcatgagccacc 148560 gtgcctagcc tattgttgta gtttttaaga ggttgtatcc ttattatgttcgtaatatct 148620 tacaaaagat taaaattaac aacaaaaaaa aagaggatat cttctctgctaatagactaa 148680 gtcaacactg cccttttgaa tcttaatctt gactaggtta ataattgtggaatttgaaag 148740 ctactcctaa atttagggta atttatatct ctttagaaat aattggtgtttttctttttg 148800 ttgggttttt tttttttttt tttttttttt ttttgagaca tagagttttgctcttgttgc 148860 ccaggctgga gtgcagtggt gcaatcttgg ttcactgcaa cctccacctctcaggttcaa 148920 gagattctcc tgcctcagcc tgctgagtat ctgggattaa cgcccggctaatttttgtat 148980 tttgtttagt agagatgggg tttcgccatg ttggccaggc tggtctacgaactcctgacc 149040 tcaggtaatc cacctacctt ggcctcccaa agtgctggaa ttacaggcatgagccaccgc 149100 gcccagccag aaataggttc ttaagcacct gtttcacatg gccacattttaataaattta 149160 cttactatga atattggaga ctccccacta tatcacaagt taaaatttaagttttactat 149220 ttagatgtag ttttttcctc ttaatttact ctacattgaa ggtttttatttcttagtatc 149280 tgaacacttt agaattaaac tctcttggag agaaacctga caattatggttctgtgcttc 149340 agtatggtca atatctacgt ctcctttatg tttcacttaa attgtgatattaaaatgaca 149400 ttaggtgggt cacatacttg gtgtaaaaaa taaaaaagaa atattaaaaatttaaaaagg 149460 tattaggaaa agttgtaagt aagattatat gacccattaa aaaaaagctaggaagttgca 149520 gacagttaat tacttgtcct gtttttgatc aaggaagtta ggttttatacacagaaggtt 149580 gatttggtgt ctgacattgg aactgaatgg agatagtact ttattagtctctggagaaaa 149640 aatcttactt tatatagtct gagagataac atatatgaat tagacagaaatatagcagat 149700 gttaaggacc aaatgagtag tatagaaaaa tgctacagtt cagagtagtgattgatcagt 149760 gaagcctgga acatcttagt gaagatgtag gacttgggct agtccttgaagaaaagggag 149820 acatttattt gtaatgtttt gcaataattt cccaccaaga agatgagacgttttttagaa 149880 ctactatgtt ggatttgtaa atggtatgca tgttaaccaa acagtgccctgggagcttag 149940 ttattcttga cataaattgg taaaaaagaa ccaagtatga attactgctaaaatttacct 150000 catttatatc atttaaaaat tatattaata tgattataag acataatgtcattaacattt 150060 taacctgtga ttaagtctta atattttggt tttattgaat cttaacaaatttcagttttt 150120 attttcagca tatactttta attactaggc ttataaactc ccagtactatattaaggact 150180 attttcagtt tatatctgat ttttttaaag aaggatgtgc atactttgtttgccttttta 150240 aaaaccctga cttttattat gtataagaat tgagcttcca ttaatgacagtttatttaaa 150300 aattgtagta agttctgtga caacttatra atgtcataaa gaacatgtagttttggattg 150360 ttctatgttt ctaaaatgtg gaattaattt atacttaggg aatgttggattttattttgt 150420 gttacttaat tcctttccct tcatagcctg tacaggtcag aaaagagtgaaagatgctca 150480 gggaggagga agttcatcaa aaaagcagaa aagaagccat aaagcaacagtggtaaacaa 150540 caaaaagaag ggaaaaggca gtaagtgtga aatctctaat ttttaaaatataaaaataat 150600 agctgataat tttaccccca gtaagaaaat ggtattcagg gtatgggatagtacactatt 150660 ttgattttgt ctgtacactc aaaaaaagtc acacaaattc tgtaaggctacttgctttaa 150720 aaaacaaaat agacaaaaaa aaaacatgca ctaggacaaa tacctaatgtaaatgatgag 150780 ttgatgggtg cagcaaacca acatggcaca tgtataccta tgtaacaaacctgcacattg 150840 tgcacacgta ccctagaact taaagtataa taataaaaag aaaaaagaatttctgcaaaa 150900 acaaacaaaa caaaacaaaa aaaacacagg ttttctaatc ttaggtaaattctagtttta 150960 agcaagttgg tgttattcag cggtggtatt gattgctgat gaaaaatcaagtaatctgtt 151020 tttgaaataa tagccaatat attataaaca tcaacatatt tgttacctttgtattccaca 151080 gttcttttca cttgctataa aatgtatcac acattgtgaa atatttcaactacatgttat 151140 ttttattaca gtgaaattga ttgcttagta attcttcaag gcaaaatcagcatagaagta 151200 taacaatcaa gataacttta gaactataat tccaacaact ccagtctacaacatttgtct 151260 ttggtactct atattatgtg acctgagagt aactacaaac aatactttttgctaagtatg 151320 ctccaacttt agcaacgact ccgtgcatca atccacagaa aatatatatactgtatttct 151380 tctgagggct gcacatttta tctctctttt gtagacaaag taagaagcagaaaatatgta 151440 aagaattttt tttcaggtgc cccacactga cctcctgctg ctcttccaggagactcctgc 151500 cacctccact gcctgacctt tgctagcagc cggcttggct gcacttcagtgcagaaaagg 151560 gttattcggc cagctccccg aggttctgct gagcccatat gacttccaggcggtgaatat 151620 ggcgtccctg gggccccggg cggctgtgct cagcaaggcc atgaaagccaggctgagaca 151680 gtccactgca tagagtgggg ccaagccttg ggtggcttag ctaggagttgccaggggttc 151740 cagagcagca gtgggggagc tgggagggaa tttgtgtata tgtgtcaaccagtggtggag 151800 tttctttatt cccaaaattt cacagtggga ggagttgctg ctatctacctttactatttg 151860 gaataatttc tggctttgag agtttaactc cttttttttt tttttttttttttttttttt 151920 tggtgatgtt gttaagtgaa aaatcagtaa atatatgcca aatcagtcagtcatcaattg 151980 tttggtgtaa ctggtaggat atttaaagtg ttttttcttt cactgtgatgtttttgtctt 152040 caagagttta ctatttaaat gacatttcct aagagagcgc atcttcaatgagttatttag 152100 tatattcata taatacagag atatagtgtt ctcccatttt acttattgggattctgccat 152160 ggtaaatcag aatgagttaa ccattcaaga gaataattta gtaacatgaattaattctga 152220 tggaatctaa actaatactt tgtatccaga aagaggttat ctatggaaatactaatccca 152280 tcacctacct gttcacatgg taagctgtca aggtcaagta tttccattttagtgctgaag 152340 tttaattagc aatagcagtt gtaacataat tgttacacag acttctgaattgttcataaa 152400 atactgaata ttttattagg gtgaaactga ttattttagt caacccacagcataggagtt 152460 atttaagagt aatttcatta tacatatcaa acttcagagt ttattcccgaggagttcttt 152520 attgaatatg atgattgtgc atcattattt gtttgtggcc aaaggagcaggaaaatgttg 152580 catataccca atgttacata ttcattgtag aggtttctac aattaatcattttaaaagat 152640 gatatatttt atgtattaac ataatagagt aaaaagccat tcagatgattactgtatatt 152700 tgacagtcta ccaagcataa tgatagatta gtgtgagtga ttttaaaagtatacatatta 152760 ttggcagttc aggtaagaag aattttttgt ttttgctttt cctaagatgctgctgcattt 152820 gtatatgatt ttccaggttt ctaggcaggt tgttttctgt aggactaaattcaaatggct 152880 aattttaaat tacctactaa aatctgtgac aaatttattg ttacatttttgtttattaaa 152940 tcttttcttt ctctttcagc aaatagtagt gatagtgaag aactttcagctggtgaaagt 153000 ataactaaga gtcagccagt caaatcagtt tccactggaa tgaagtctcatagtaccaaa 153060 tctcccgcaa ggacgcagtc tccaggaaaa tgtggaaaga atggtgataaggatcctgat 153120 ctcaaggaac ccagtaatcg attacccaaa gtttacaaat ggagttttcagatgtgtaag 153180 tgacatgtta aattgacaag catacaaact tcatcctagt aactctttttgttttatttt 153240 gtttttgttt ttagagacag agtctcgctc tgtcaccagg ctggagtgcggtggtgcgat 153300 cttggctcac tgtaatcttc agcctcctgg gttcaagcca tcctcctgcctcagcctccc 153360 aagtagctgg gattacaggc acgcgccacc acacccagct aatttttttgtgtgtttagt 153420 agagacaggg tttcaccatg ttggccagga tggtctccat ctcctgacctcatgatccgc 153480 ccgcctcggc ctcccaaagt gctgggatta caggcatgag ccactgcgcctggccttttt 153540 ttttgagaca gagtctctct gtcgcccagg ctagagtgca gtgcagtggcatagtctctg 153600 ctcactgcaa cctccatctc cctggttcaa atgattctcc tgcctcaacctcccgagtag 153660 ttgggattac agacgcccac caccacacct ggctaatttt tttgttgttgttgtattttt 153720 agtagacatg gggtttcacc atgttggcca ggctggtctt gaactcctgacctcaggtga 153780 tccatccgcc tctgcctcgc aaagtgctgg gattttgggc atgagccaccatgcccagcc 153840 ccaatcctag taactcttca tgccaatact ctgaaaaaga ggctttaccaaacttaatag 153900 atgtactaat gtacaatgta tagaccttat ttggatcccg gtttgaataaataaattggt 153960 taaagaaaaa ttttaaggca gttgaggcaa atgccaacac tgactagatatttctgatat 154020 ggctgggcgc agtggctcat gcctgtaatc ccagcacttt gggaggctgaggtgggtgga 154080 tcacgaggtc aggagttcga gaccagcctg accagcatgg tgaaaccctgtctctactaa 154140 taaaacaaaa aattagctgc gcgtggtggc acgcgcttgt agtcccagctactcaggagg 154200 ctgaggcagg agaattgctt gcacctggca ggtggaggtt gcagtgagccgaggttgcgc 154260 cactgcactc cagcctgggc gacagagtga gaccccatcc cagaaaaaaaaaaaaagata 154320 cttctgatat gatgtcggta atttgattta aaagtatctg gtgttaggggtgaggaatgg 154380 ataggggtgc agatgataca ggttttgcca taaattgatg attactgaagctggataatg 154440 agtacatggg gttcactatc cttctctcta tacttttgta tatgtgagaaatcttcataa 154500 atcttcattt aaaaaaaggt atatatatat atgttttagg tatacatatatatatatata 154560 tatatatata tatatatata tatatttttt tttttttttt ttttttttttttaaatagag 154620 acgaggtctc attatgttgc ccaggctaat cttgaactcc tgagctcaagactgagctga 154680 tccttccacc tctacttccc aaagtgctag gattacaggt gtgagccaccacacccagcc 154740 aatatgtata tttttttaat actactctag agtttttcac acaaggaaataccttaagta 154800 ttcttaggag attgaagatt gctttagagc tttttaaaat tgccttctaatttaaatttt 154860 tacacactct ttaaaaaaac ctaaaaaata acagagaaca gaagagaaaaatttatccac 154920 agtcttgtta ctcacaaaat gtgtacaatt tagcattttg gtgtctttccaggttttttg 154980 ttttgttttg tttttgtgtt tttgttattt ttagacagag tctcactctgttgcccaggc 155040 tagcgtgcag tggcacaatc tcggctcact ccaacctcca ccttccagattcaagcgatt 155100 atcctgcccc agcctcccga gtagctggga ttgcaggcac ccgccaccatgcccagctaa 155160 tttttgtagt tttagtagag acagggtttc accatcttgg ccaggctggtatcgaactcc 155220 tgacctcgtg atccgcccac ctcggcctcc caaagtgcta ggattacaggtgtgagccac 155280 cgtgcccagc cccagtttta tttttaagtg tgatttttta ctgtggtaatactgtatatg 155340 gatgtggata tatgtagatc ttaaggtgtt taatgctgta catattcattcaacaaatac 155400 tgtgcattta ttatgtgcca ggcattgttc taggctagat aaaaaatttggaaaacaaat 155460 atttcagaag ccttagtttt ttagttcatc tgcctcaacc ttattcagcagccatgctcg 155520 atgttctctc ctttttgtat gttaaaattt tctttaaaaa tgtctgttaatgaaagcttt 155580 aaatttatag cggacctgga aaatatgaca agtgccgaac gcatcacaattcttcaagaa 155640 aaacttcaag aaatcagaaa acattatctg tcattaaaat ctgaagtagcttccattgat 155700 cggaggagaa agcgtttaaa gaagaaagag agagaaagta agtatttttactttattttt 155760 atttatttat ttattttgag acagagtttc actcttgtcg ccaggctggagtgcaatggc 155820 gcaatctcgg ctcactgcaa cctccacctc ctgggttcaa acagttctcctacctcagct 155880 tcctaagtag ctggaattac aggcatgcac caccaagccc ggctaattttgtatttttta 155940 ggtagagaca gggtttcgcc atgtcaatca ggctggtctc taactcctgacctcaggtga 156000 tctacctgtc ttggcctccc aaagtgctaa gattacaggc gtgagccaagagtgccctgc 156060 cagtattttt actttattta aacataaacc agaatttctc actctgcagttagactgcca 156120 tgactttgtc tattttcagg caaattcttt aatttcttta tcttattttcctcatctcta 156180 aagtgaaatt atctcaaata aaaaaattat ttcagatcat aatattcactttcatagagt 156240 ttatactcta ccaaaaacat cctaataaga taatttcaga tattgaaagcactatgaaga 156300 aaatgatgtt aaggtagtga ttggatgggt tactttagat tacaaatggtcagtatattt 156360 ttgttgatac ctgaatgaca tgaggaagtg agataaatta aaatctgggaggaggccggg 156420 cacagtggct catgcctgta atcccagcac tttgggaggc tgcggtgggtggattgcctg 156480 aggtcaggag gtcgagacca gcttggccaa catagtgaaa ccccgtctctactaaaaaat 156540 acaaaaaatt agctgtgcgc ggtggcgggt gcctgtaatc ccagctacttgggaggccga 156600 cgcaggagaa ttgcttaaac ccaggaggca gatgttgcag tgaacggagatcatgccatt 156660 gcactccagc ctgggcaaca agagctaaac tctgtctcaa aaaaaaaaagacatctatcc 156720 agaaactcct tctaaacaat gttttgtaaa tatagtcacc acaaattctttataatgaat 156780 gattttgcta aatagagcct ctctactggg ttagcattaa aagtcggttcctaaatacta 156840 ttttaagaaa aatccatagg aaaatgctta tcctggttac caaagaaatgcaaatcaaat 156900 aaggtatcat tctttttttt tggagatgga gttttgctct gtcgcccaggctggagtgca 156960 gtggcgcgat ctcagctcac tacaacctcc gcctcctgag ttcaagcgattatcctgcct 157020 cagtctcccg agtggctggg attacaagcg tgctgccacg cccagctaattttttatttt 157080 tagtagagat ggggtttcac catgttggcc aggatgcagg atggctcgatctcttgactt 157140 cgtgatccgc ctgcttcagt ctcccaaagt gctgggatta caggcatgagccactgcgac 157200 tggcccaaat aaggtatcat tcttaccaaa aaaattaaaa ctaaaaccaatgcaggaaca 157260 gtgtagtgaa atatataagt tatgagttgt aatatagtag aatattactcaactttgaaa 157320 agaaaaggat ctgtatgtac tgatatggaa caatctctta aaatatattgtttaaaaaaa 157380 agtcagacac tgaactatgc ttccacttgt gtgtgtgtgg tttttttttttttttttttt 157440 ttttgagacg gagtctcggt cagtcaccag gctggagtgc agtggcgcgatcttggctca 157500 ctgcaacctc tgccttccgg ttcaagcgat tctcctgcct cagcctcccgggtagctagg 157560 actacaggtg cgtgccgcca tgcccagcta atttttgtaa tattattattaatttttgag 157620 acagagtatc tctctgtcat ccaggctgga gtgtagtggt gcaatcttggctcactgcaa 157680 ctccgcctcc cgggttcacg ccattctcct gcctcagcct ctctagtagctgggactaca 157740 ggcgcccacc accacatctg gctaattttt tgtattttta gtagagactgagtttcattg 157800 tgttagccag gatggtctcg atctcctgac cttgtgatcc gcccacctagtcctcccaaa 157860 gtgctgggat tacaggtgtg agccaccgtg cccggcctcc acttatgtttttaaaggtgt 157920 tgctatatct atatatttta aatttgcata tcatatctct agattctagagataaaattc 157980 ctgtagaaca ggggttgcaa acattttctg taaagagaca ataaatatgttaggctttgt 158040 gggccatgtg gtctctgtag aaactactta tctctgccat ggtaagtgtgaaaactccca 158100 taggcaatat gtaaacaaat aggcatggct gtgttccagt acaatttttctttccaaaga 158160 caagtaagcc agatttgccc ctggggtagt ttttttgcca gccttttttctagagttgta 158220 atgaatatga atcaactggt agcaatgggg aagggaattt gggtgattaggaggttcctg 158280 gatgagagtg agatttgctt ttctatccta ttacccctgt actgaatgaattttttaaat 158340 ctgtgcatgt atttaaaaat attaatttat gaacactaat ttataacagagaggccgggc 158400 gcaattgctc acgcctgtaa ttccagcact tagggaagcc aaggcaggcagatcacctga 158460 ggtcaggagt tcgagaccag actggccaac atgacgaaac cccatctctactaaaaatag 158520 aaacattagc cggacatggt ggcgcatgcc tgtaatccca gatactcaggaggctgagac 158580 aggaaaattg cttgaaccca ggaggcaaag gttgcagtga gctgagattgcactgctgca 158640 gtccagcctg ggcaacagag caagaccccc atctcaaagg aaaaaaaaaaaaaaggaggc 158700 tgaggcagga gaatggcgtg aacctggaag gcggagcttg cagtgagccgagatcgcacc 158760 actgcactcc agcctgggcg acagagtgag actccgtctc aaaaaaacaaacaggctggg 158820 cgcggtggct cacacctgta atcccggcat tttgggaggc tgagatgggcggatcatgag 158880 gtcaggagat cgagaccatc ctggctaaca tggtgaaacc ccatctctactaaaaataca 158940 aaaaaattag ccgggcgtgg tggcgggcgc ttatagtccc agctactgggggggctgagg 159000 caggagaatg gcgtgaactc aggaggcaga acttttagtg agccaagatcgagccactgc 159060 actccagcct gggcgacaga cagagcgaga ctttgtctca aaaaacaaacaaacaaaaaa 159120 aaacgaaaaa aggaaacagt gagagacttc ctctgatgaa aataactaatgtgttatttg 159180 ctttgtaaac ctagtggcgg agaatgcaac agttgagcta tgcactgttttgatccaact 159240 tgaagaagca attaagcctc ccactcttgt caccatttac atgtacaagaaaactcctaa 159300 gtacagaaag atggagggat taggagggga caaatgattt ttggatggattgcagatttt 159360 tcctgttttg atacttgttt cactgttaca aaaagtgtat tctgtattttatttctgtgt 159420 cttgtagact aggcacagtt ctttccctct ttgacccacg caggagccttccctggtctg 159480 tccttttcat tacttctgta gttgggcact gtctagcttc ttgagctacactagcttttc 159540 ctttcttcac atcgtagaac tgtgagaatt gaccactgtt gttgtaagctaaatgatttt 159600 ggacaatata gcggaatttt agttcagagg actagtattt tctgtctattgttagacata 159660 aatttttatt aagctcttgg tttggtctcc cttttcctta ggtgctgctacatcctcatc 159720 ctcctcttca ccttcatcca gttccataac agctgctgtt atgttaactttagctgaacc 159780 gtcaatgtcc agcgcatcac aaaatggaat gtcagttgag tgcaggtgacagcaggactt 159840 gctaaagcac tttgcactta atggctgttg agggccactt tttttttatactgcacagtg 159900 gcacaaaaaa atatcagaca agcactattt tatatttaaa aattgtttcttgacaagctg 159960 acttggcact taagtgcact tttttatgaa gaaaaagtac aatgaactgcttttcctcaa 160020 gcaataattg kttccaactt gtctgggaat tgtgtgtctg gtaactggaaggccttccac 160080 tgtggcaaat ggaggctttt cactgcctgt agagacaata cagtaagcatagttaagggg 160140 tgggtcagaa catgttaaga taacttactg tatatgtatt cccttgtattttgttaaagc 160200 tggaacattt gatatttttc catttattta tgaaaaaata tgaacctattttcatttgta 160260 caaggtaatt gttttttaaa gcaagtcacc ttagggtggc tttaattgtataagtcaagc 160320 acatgtaata aattcaaaac ctgcagttaa caggatatta gacatcaatcctggtaacca 160380 aatattaaag attctcttta aaaaagactg aacatgttta caggtttgaattaggctaaa 160440 aggtcttgca gtggcttttc atggcccttc aaattggaat ggaactactgtactttgcca 160500 tttttctata aatcagtatt tttttttaat tttgatatac attgtgtgaaaaaagaaaat 160560 ggctaataaa ctgtattaaa tcttaaacaa tgtataaaga ttgtacttagccagttcaaa 160620 gtgtatattt attcataatg aattataaca gttatatttt tgtgttttcttgtaaatgtt 160680 tcttttccct taaatacaga taattcattt gtattgctta ttttattatgagctacaaca 160740 aaaggacttc aggaacaagt aatgtattag tatggttcaa gattgttgataggaactgtc 160800 tcaaaaggat ggtggttatt ttaaatataa atagctaatg ggggtggtaggcctataaaa 160860 ttaaatgcct tgtataaaat ccaaaatgaa tgcaaaattg ttttcacttgtattgacttt 160920 atgttgtatg attccaatct ctgttctgtt tggcacttgt atttaattcttcacctttgt 160980 aagacatttg tatattgtgg atgtgttcat tcaagctatt taatatctggcactgttaat 161040 acacagtact ttattgtaca gactgtttta ctgttttaat tgtagttctgtgtacttttt 161100 ttggatgggg ctggcatgtt ttctttgttt cctggcaata cgacgtgggaatttcaatgc 161160 gttttgttgt agatgctaac gtgtcagaat cctttacatt caacttttctaagaaaagca 161220 ttttcagtct tgtagtgtgt gcttacagta actaattttg ttgaaaatggtttcaagtta 161280 ttcaaatttg tacaggactg taaagatttg ttgacagcaa aatgttgaagaaaaaagctt 161340 atagaataaa agctataaag tatatattag gatctgcaaa caatgaagaattatgtaata 161400 tattgtacaa atgtaagcaa aggctctgaa ataaaatgcc atagtttgtgaatccttgat 161460 ttttgtttct aaaagattta gtaattttag ttcatttctg tatgtgatgactgactggaa 161520 catacatatc cagcacgtat tatcacaggg gattaattga tacacaaaaaaaggaagatt 161580 ctacctatga aaattaaaag tccattaatc agataaggaa tgtattaggcattctttttt 161640 tttttttttt ttttggacac ggagtcttgc tctgtcaccc aggctagagtgcagtggcgc 161700 aatctccagc tcactgcaac ctctatctcc cgggttcaag caattctccagcttcagcct 161760 tccgagtagc tgggattata gacatctgcc agcactcctg gctaatttttgtatttttag 161820 cagagacggg gtttcaccgt actggtcagg ctggtctcaa actcctgacctcatgtgatc 161880 cacccacctt ggcctcccaa tgtgctggga ttacaggcgg gagccaacacacccagccta 161940 ggcattcttt tatctttgca cacactattt tgcttgagtc tgaatttaaatatttttctt 162000 atcacttgaa gaattgtcca aatttgaaaa ttaagtgttt tttttaaaatttatttaaca 162060 cttgaaacca ttaccagcgg ctttttaaaa tttttaattt agttagacctttccgggtct 162120 tttatacttc agtgtgttct attgcacatt gcaatcatct ggacattgttaaaagtatat 162180 tcagtactca caccccactc ccaaggagtt atatttaatt ggttgggggtagtacctgga 162240 tgttgatctt taatttttaa aggtctctag tgatattaat atgcatctgggttgagaaac 162300 actgctttgc cgcaaacttc taaaaatcta taatctagtt ttttggccccacttattgga 162360 ctttctacca acagaaaacc tttcttggct gggcgcagtg gctcaacgcctgtaatccta 162420 gcactttggg aggccgaggc aggcggatca 162450 2 273 DNA Homosapiens CAAT_signal 139..147 AACCAATCC 2 ccccagagta tggactttatttcccagaaa gccttgaggc gtaactttct gtttccatag 60 aactggtggg aaaatggcgtcgttgtttgt atccagggac caataggaac agtgtatagg 120 cgggttctaa agaactttaaccaatccaag gtcgtctaag aggccatccg ggaaagaggt 180 aggggagggg gggaaaaaaaatctagggga ggggagaaag ggggggaacc tagagtcggt 240 gggggggaag cgatgtttgcccgtcagtcg agt 273 3 999 DNA Homo sapiens 3 atccttgatt tttgtttctaaaagatttag taattttagt tcatttctgt atgtgatgac 60 tgactggaac atacatatccagcacgtatt atcacagggg attaattgat acacaaaaaa 120 aggaagattc tacctatgaaaattaaaagt ccattaatca gataaggaat gtattaggca 180 ttcttttttt tttttttttttttggacacg gagtcttgct ctgtcaccca ggctagagtg 240 cagtggcgca atctccagctcactgcaacc tctatctccc gggttcaagc aattctccag 300 cttcagcctt ccgagtagctgggattatag acatctgcca gcactcctgg ctaatttttg 360 tatttttagc agagacggggtttcaccgta ctggtcaggc tggtctcaaa ctcctgacct 420 catgtgatcc acccaccttggcctcccaat gtgctgggat tacaggcggg agccaacaca 480 cccagcctag gcattcttttatctttgcac acactatttt gcttgagtct gaatttaaat 540 atttttctta tcacttgaagaattgtccaa atttgaaaat taagtgtttt ttttaaaatt 600 tatttaacac ttgaaaccattaccagcggc tttttaaaat ttttaattta gttagacctt 660 tccgggtctt ttatacttcagtgtgttcta ttgcacattg caatcatctg gacattgtta 720 aaagtatatt cagtactcacaccccactcc caaggagtta tatttaattg gttgggggta 780 gtacctggat gttgatctttaatttttaaa ggtctctagt gatattaata tgcatctggg 840 ttgagaaaca ctgctttgccgcaaacttct aaaaatctat aatctagttt tttggcccca 900 cttattggac tttctaccaacagaaaacct ttcttggctg ggcgcagtgg ctcaacgcct 960 gtaatcctag cactttgggaggccgaggca ggcggatca 999 4 6002 DNA Homo sapiens allele 1319 5-130-257polymorphic base A or G 4 ccggagtgag gagctcggtc gccgaagcgg agggagactcttgagcttca tcttgccgcc 60 gccacggcca ccgcctggac ctttgcccgg agggagctgcagagggtcca tcgccgccgt 120 cctctggagg gcagcgcgat tgggggcccg gacctccagtccggggggga tttttcgtcg 180 tccccctccc cccaaccagg gagcccgagc ggccgccaaacaaaggtacc agtcgccgcc 240 gcgggaggag gaggagccgg agcctctgcc tcagcagccgctggacccgc cgcccttctt 300 ccccatctct cccccgggcc tgctggtttt gggggggagaaggagagagg ggactctgga 360 cgtgccaggg tcagatctcg cctccgagga aggtgcagctgaacctggtg ttttagagga 420 taccttggtc ccagagtcat c atg aag gcc ctt gatgag cct ccc tat ttg 471 Met Lys Ala Leu Asp Glu Pro Pro Tyr Leu 1 5 10aca gtg ggc act gat gtg agt gct aaa tac aga gga gcc ttt tgt gaa 519 ThrVal Gly Thr Asp Val Ser Ala Lys Tyr Arg Gly Ala Phe Cys Glu 15 20 25 gccaag atc aag aca gca aaa aga ctt gtc aaa gtc aag gtg aca ttt 567 Ala LysIle Lys Thr Ala Lys Arg Leu Val Lys Val Lys Val Thr Phe 30 35 40 aga catgat tct tca aca gtg gaa gtt cag gat gac cac ata aag ggc 615 Arg His AspSer Ser Thr Val Glu Val Gln Asp Asp His Ile Lys Gly 45 50 55 cca cta aaggta gga gct att gtg gaa gtg aag aat ctt gat ggt gca 663 Pro Leu Lys ValGly Ala Ile Val Glu Val Lys Asn Leu Asp Gly Ala 60 65 70 tat cag gaa gctgtt atc aat aaa cta aca gat gcg agt tgg tac act 711 Tyr Gln Glu Ala ValIle Asn Lys Leu Thr Asp Ala Ser Trp Tyr Thr 75 80 85 90 gta gtt ttt gatgac gga gat gag aag aca ctg aga cga tct tca ctg 759 Val Val Phe Asp AspGly Asp Glu Lys Thr Leu Arg Arg Ser Ser Leu 95 100 105 tgc ctg aaa ggagag agg cat ttt gct gaa agt gaa aca tta gac cag 807 Cys Leu Lys Gly GluArg His Phe Ala Glu Ser Glu Thr Leu Asp Gln 110 115 120 ctc cca ctc accaac cct gag cat ttt ggc act cca gtc ata gga aag 855 Leu Pro Leu Thr AsnPro Glu His Phe Gly Thr Pro Val Ile Gly Lys 125 130 135 aaa aca aat agagga aga aga tct aat cat ata cca gag gaa gag tct 903 Lys Thr Asn Arg GlyArg Arg Ser Asn His Ile Pro Glu Glu Glu Ser 140 145 150 tca tca tcc tccagt gat gaa gat gag gat gat agg aaa cag att gat 951 Ser Ser Ser Ser SerAsp Glu Asp Glu Asp Asp Arg Lys Gln Ile Asp 155 160 165 170 gag cta ctaggc aaa gtt gta tgt gta gat tac att agt ttg gat aaa 999 Glu Leu Leu GlyLys Val Val Cys Val Asp Tyr Ile Ser Leu Asp Lys 175 180 185 aag aaa gcactg tgg ttt cct gca ttg gtg gtt tgt cct gat tgt agt 1047 Lys Lys Ala LeuTrp Phe Pro Ala Leu Val Val Cys Pro Asp Cys Ser 190 195 200 gat gag attgct gta aaa aag gac aat att ctt gtt cga tct ttc aaa 1095 Asp Glu Ile AlaVal Lys Lys Asp Asn Ile Leu Val Arg Ser Phe Lys 205 210 215 gat gga aaattt act tca gtt cca aga aaa gat gtc cat gaa att act 1143 Asp Gly Lys PheThr Ser Val Pro Arg Lys Asp Val His Glu Ile Thr 220 225 230 agt gac actgca cca aag cct gat gct gtt tta aag caa gcc ttt gaa 1191 Ser Asp Thr AlaPro Lys Pro Asp Ala Val Leu Lys Gln Ala Phe Glu 235 240 245 250 cag gcactt gaa ttt cac aaa agt aga act att cct gct aac tgg aag 1239 Gln Ala LeuGlu Phe His Lys Ser Arg Thr Ile Pro Ala Asn Trp Lys 255 260 265 act gaattg aaa gaa gat agc tct agc agt gaa gca gag gaa gaa gag 1287 Thr Glu LeuLys Glu Asp Ser Ser Ser Ser Glu Ala Glu Glu Glu Glu 270 275 280 gag gaggaa gat gat gaa aaa gaa aag gag grt aat agc agt gaa gaa 1335 Glu Glu GluAsp Asp Glu Lys Glu Lys Glu Xaa Asn Ser Ser Glu Glu 285 290 295 gar gaagaa ata gaa cca ttt cca gaa gaa agg gag aac ttt ctt cag 1383 Glu Glu GluIle Glu Pro Phe Pro Glu Glu Arg Glu Asn Phe Leu Gln 300 305 310 caa ttgtac aaa ttt atg gaa gat aga ggt aca cct att aac aaa cga 1431 Gln Leu TyrLys Phe Met Glu Asp Arg Gly Thr Pro Ile Asn Lys Arg 315 320 325 330 cctgta ctt gga tat cga aat ttg aat ctc ttt aag tta ttc aga ctt 1479 Pro ValLeu Gly Tyr Arg Asn Leu Asn Leu Phe Lys Leu Phe Arg Leu 335 340 345 gtacac aaa ctt gga gga ttt gat aat att gaa agt gga gct gtt tgg 1527 Val HisLys Leu Gly Gly Phe Asp Asn Ile Glu Ser Gly Ala Val Trp 350 355 360 aaacaa gtc tac caa gat ctt gga atc cct gtc tta aat tca gct gca 1575 Lys GlnVal Tyr Gln Asp Leu Gly Ile Pro Val Leu Asn Ser Ala Ala 365 370 375 ggatac aat gtt aaa tgt gct tat aaa aaa tac tta tat ggt ttt gag 1623 Gly TyrAsn Val Lys Cys Ala Tyr Lys Lys Tyr Leu Tyr Gly Phe Glu 380 385 390 gagtac tgt aga tca gcc aac att gaa ttt cag atg gca ttg cca gag 1671 Glu TyrCys Arg Ser Ala Asn Ile Glu Phe Gln Met Ala Leu Pro Glu 395 400 405 410aaa gtt gtt aac aag caa tgt aag gag tgt gaa aat gta aaa gaa ata 1719 LysVal Val Asn Lys Gln Cys Lys Glu Cys Glu Asn Val Lys Glu Ile 415 420 425aaa gtt aag gag gaa aat gaa aca gag atc aaa gaa ata aag atg gag 1767 LysVal Lys Glu Glu Asn Glu Thr Glu Ile Lys Glu Ile Lys Met Glu 430 435 440gag gag agg aat ata ata cca aga gaa gaa aag cct att gag gat gaa 1815 GluGlu Arg Asn Ile Ile Pro Arg Glu Glu Lys Pro Ile Glu Asp Glu 445 450 455att gaa aga aaa gaa aat att aag ccc tct ctg gga agt aaa aag aat 1863 IleGlu Arg Lys Glu Asn Ile Lys Pro Ser Leu Gly Ser Lys Lys Asn 460 465 470tta tta gaa tct ata cct aca cat tct gat cag gaa aaa gaa gtt aac 1911 LeuLeu Glu Ser Ile Pro Thr His Ser Asp Gln Glu Lys Glu Val Asn 475 480 485490 att aaa aaa cca gaa gac aat gaa aat ctg gay gac aaa gat gat gac 1959Ile Lys Lys Pro Glu Asp Asn Glu Asn Leu Asp Asp Lys Asp Asp Asp 495 500505 aca act agg gta gat gaa tcc ctc aac ata aag gta gaa gct gag gaa 2007Thr Thr Arg Val Asp Glu Ser Leu Asn Ile Lys Val Glu Ala Glu Glu 510 515520 gaa aaa gca aaa tct gga gat gaa acg aat aaa gaa gaa gat gaa gat 2055Glu Lys Ala Lys Ser Gly Asp Glu Thr Asn Lys Glu Glu Asp Glu Asp 525 530535 gat gaa gaa gca gaa gag gag gag gag gag gaa gaa gaa gaa gag gat 2103Asp Glu Glu Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp 540 545550 gaa gat gat gat gac aac aat gag gaa gag gag ttt gag tgc tat cca 2151Glu Asp Asp Asp Asp Asn Asn Glu Glu Glu Glu Phe Glu Cys Tyr Pro 555 560565 570 cca ggc atg aaa gtc caa gtg cgg tat gga cga ggg aaa aat caa aaa2199 Pro Gly Met Lys Val Gln Val Arg Tyr Gly Arg Gly Lys Asn Gln Lys 575580 585 atg tat gaa gct agt att aaa gat tct gat gtt gaa ggt gga gag gtc2247 Met Tyr Glu Ala Ser Ile Lys Asp Ser Asp Val Glu Gly Gly Glu Val 590595 600 ctt tac ttg gtg cat tac tgc gga tgg aat gtg aga tac gat gaa tgg2295 Leu Tyr Leu Val His Tyr Cys Gly Trp Asn Val Arg Tyr Asp Glu Trp 605610 615 att aaa gca gat aaa ata gta aga cct gct gat aaa aat gtg cca aag2343 Ile Lys Ala Asp Lys Ile Val Arg Pro Ala Asp Lys Asn Val Pro Lys 620625 630 ata aaa cat cgg aag aaa ata aag aat aaa tta gac aaa gaa aaa gac2391 Ile Lys His Arg Lys Lys Ile Lys Asn Lys Leu Asp Lys Glu Lys Asp 635640 645 650 aaa gat gaa aaa tac tct cca aaa aac tgt aaa ctt cgg cgc ttgtcc 2439 Lys Asp Glu Lys Tyr Ser Pro Lys Asn Cys Lys Leu Arg Arg Leu Ser655 660 665 aaa cca cca ttt cag aca aat cca tct cct gaa atg gta tcc aaactg 2487 Lys Pro Pro Phe Gln Thr Asn Pro Ser Pro Glu Met Val Ser Lys Leu670 675 680 gat ctc act gat gcc aaa aac tct gat act gct cat att aag tccata 2535 Asp Leu Thr Asp Ala Lys Asn Ser Asp Thr Ala His Ile Lys Ser Ile685 690 695 gaa att act tcg atc ctt aat gga ctt caa gct tct gaa agt tctgct 2583 Glu Ile Thr Ser Ile Leu Asn Gly Leu Gln Ala Ser Glu Ser Ser Ala700 705 710 gaa gac agt gag cag gaa gat gag aga ggt gct caa gac atg gataat 2631 Glu Asp Ser Glu Gln Glu Asp Glu Arg Gly Ala Gln Asp Met Asp Asn715 720 725 730 aat ggc aaa gag gaa tct aag att gat cat ttg acc aac aacaga aat 2679 Asn Gly Lys Glu Glu Ser Lys Ile Asp His Leu Thr Asn Asn ArgAsn 735 740 745 gat ctt att tca aag gag gaa cag aac agt tca tct ttg ctagaa gaa 2727 Asp Leu Ile Ser Lys Glu Glu Gln Asn Ser Ser Ser Leu Leu GluGlu 750 755 760 aac aaa gtt cat gca gat ttg gta ata tcc aaa cca gtg tcaaaa tct 2775 Asn Lys Val His Ala Asp Leu Val Ile Ser Lys Pro Val Ser LysSer 765 770 775 cca gaa aga tta agg aaa gat ata gaa gta tta tcc gaa gatact gat 2823 Pro Glu Arg Leu Arg Lys Asp Ile Glu Val Leu Ser Glu Asp ThrAsp 780 785 790 tat gaa gaa gat gaa gtc aca aaa aag aga aag gat gtc aagaag gac 2871 Tyr Glu Glu Asp Glu Val Thr Lys Lys Arg Lys Asp Val Lys LysAsp 795 800 805 810 aca aca gat aaa tct tca aaa cca caa ata aaa cgt ggtaaa aga agg 2919 Thr Thr Asp Lys Ser Ser Lys Pro Gln Ile Lys Arg Gly LysArg Arg 815 820 825 tat tgc aat aca gaa gag tgt cta aaa act gga tca cctggc aaa aag 2967 Tyr Cys Asn Thr Glu Glu Cys Leu Lys Thr Gly Ser Pro GlyLys Lys 830 835 840 gaa gag aag gcc aag aac aaa gaa tca ctt tgc atg gaaaac agt agc 3015 Glu Glu Lys Ala Lys Asn Lys Glu Ser Leu Cys Met Glu AsnSer Ser 845 850 855 aac agc tct tca gat gaa gat gaa gaa gaa aca aaa gcaaag atg aca 3063 Asn Ser Ser Ser Asp Glu Asp Glu Glu Glu Thr Lys Ala LysMet Thr 860 865 870 cca act aag aaa tac aat ggt ttg gag gaa aaa aga aaatct cta cgg 3111 Pro Thr Lys Lys Tyr Asn Gly Leu Glu Glu Lys Arg Lys SerLeu Arg 875 880 885 890 aca act ggt ttc tat tca gga ttt tca gaa gtg gcagaa aaa agg att 3159 Thr Thr Gly Phe Tyr Ser Gly Phe Ser Glu Val Ala GluLys Arg Ile 895 900 905 aaa ctt tta aat aac tct gat gaa aga ctt caa aacagc agg gcc aaa 3207 Lys Leu Leu Asn Asn Ser Asp Glu Arg Leu Gln Asn SerArg Ala Lys 910 915 920 gat cga aaa gat gtc tgg tca agt att cag gga cagtgg cct aaa aaa 3255 Asp Arg Lys Asp Val Trp Ser Ser Ile Gln Gly Gln TrpPro Lys Lys 925 930 935 acg ctg aaa gag ctt ttt tca gac tct gat act gaggct gca gct tcc 3303 Thr Leu Lys Glu Leu Phe Ser Asp Ser Asp Thr Glu AlaAla Ala Ser 940 945 950 cca ccg cat cct gcc cca gag gag grg gtg gca gaggag tca mtg cag 3351 Pro Pro His Pro Ala Pro Glu Glu Xaa Val Ala Glu GluSer Xaa Gln 955 960 965 970 act gtg gct gaa gag gag agt tgt tca ccc agtgta gaa cta gaa aaa 3399 Thr Val Ala Glu Glu Glu Ser Cys Ser Pro Ser ValGlu Leu Glu Lys 975 980 985 cca cct cca gtc aat gtc gat agt aaa ccc attgaa gaa aaa aca gta 3447 Pro Pro Pro Val Asn Val Asp Ser Lys Pro Ile GluGlu Lys Thr Val 990 995 1000 gag gtc aat gac aga aaa gca gaa ttt cca agtagt ggc agt aat tca 3495 Glu Val Asn Asp Arg Lys Ala Glu Phe Pro Ser SerGly Ser Asn Ser 1005 1010 1015 gtg cta aat acc cct cct act aca cct gaatcg cct tca tca gtc act 3543 Val Leu Asn Thr Pro Pro Thr Thr Pro Glu SerPro Ser Ser Val Thr 1020 1025 1030 gta aca gaa ggc agc cgg cag cag tcttct gta aca gta tca gaa cca 3591 Val Thr Glu Gly Ser Arg Gln Gln Ser SerVal Thr Val Ser Glu Pro 1035 1040 1045 1050 ctg gct cca aac caa gaa gaggtt cga agt atc aag agt gaa act gat 3639 Leu Ala Pro Asn Gln Glu Glu ValArg Ser Ile Lys Ser Glu Thr Asp 1055 1060 1065 agc aca att gag gtg gatagt gtt gct ggg gag ctc caa gac ctc cag 3687 Ser Thr Ile Glu Val Asp SerVal Ala Gly Glu Leu Gln Asp Leu Gln 1070 1075 1080 tct gaa ggg aat agctcg cca gca ggt ttt gat gcc agt gtg agc tca 3735 Ser Glu Gly Asn Ser SerPro Ala Gly Phe Asp Ala Ser Val Ser Ser 1085 1090 1095 agc agt agt aatcag cca gaa cca gaa cat cct gaa aaa gcc tgt aca 3783 Ser Ser Ser Asn GlnPro Glu Pro Glu His Pro Glu Lys Ala Cys Thr 1100 1105 1110 ggt cag aaaaga gtg aaa gat gct cag gga gga gga agt tca tca aaa 3831 Gly Gln Lys ArgVal Lys Asp Ala Gln Gly Gly Gly Ser Ser Ser Lys 1115 1120 1125 1130 aagcag aaa aga agc cat aaa gca aca gtg gta aac aac aaa aag aag 3879 Lys GlnLys Arg Ser His Lys Ala Thr Val Val Asn Asn Lys Lys Lys 1135 1140 1145gga aaa ggc aca aat agt agt gat agt gaa gaa ctt tca gct ggt gaa 3927 GlyLys Gly Thr Asn Ser Ser Asp Ser Glu Glu Leu Ser Ala Gly Glu 1150 11551160 agt ata act aag agt cag cca gtc aaa tca gtt tcc act gga atg aag3975 Ser Ile Thr Lys Ser Gln Pro Val Lys Ser Val Ser Thr Gly Met Lys1165 1170 1175 tct cat agt acc aaa tct ccc gca agg acg cag tct cca ggaaaa tgt 4023 Ser His Ser Thr Lys Ser Pro Ala Arg Thr Gln Ser Pro Gly LysCys 1180 1185 1190 gga aag aat ggt gat aag gat cct gat ctc aag gaa cccagt aat cga 4071 Gly Lys Asn Gly Asp Lys Asp Pro Asp Leu Lys Glu Pro SerAsn Arg 1195 1200 1205 1210 tta ccc aaa gtt tac aaa tgg agt ttt cag atgtcg gac ctg gaa aat 4119 Leu Pro Lys Val Tyr Lys Trp Ser Phe Gln Met SerAsp Leu Glu Asn 1215 1220 1225 atg aca agt gcc gaa cgc atc aca att cttcaa gaa aaa ctt caa gaa 4167 Met Thr Ser Ala Glu Arg Ile Thr Ile Leu GlnGlu Lys Leu Gln Glu 1230 1235 1240 atc aga aaa cat tat ctg tca tta aaatct gaa gta gct tcc att gat 4215 Ile Arg Lys His Tyr Leu Ser Leu Lys SerGlu Val Ala Ser Ile Asp 1245 1250 1255 cgg agg aga aag cgt tta aag aagaaa gag aga gaa agt gct gct aca 4263 Arg Arg Arg Lys Arg Leu Lys Lys LysGlu Arg Glu Ser Ala Ala Thr 1260 1265 1270 tcc tca tcc tcc tct tca ccttca tcc agt tcc ata aca gct gct gtt 4311 Ser Ser Ser Ser Ser Ser Pro SerSer Ser Ser Ile Thr Ala Ala Val 1275 1280 1285 1290 atg tta act tta gctgaa ccg tca atg tcc agc gca tca caa aat gga 4359 Met Leu Thr Leu Ala GluPro Ser Met Ser Ser Ala Ser Gln Asn Gly 1295 1300 1305 atg tca gtt gagtgc agg tga cagcaggact tgctaaagca ctttgcactt 4410 Met Ser Val Glu CysArg * 1310 aatggctgtt gagggccact ttttttttat actgcacagt ggcacaaaaaaatatcagac 4470 aagcactatt ttatatttaa aaattgtttc ttgacaagct gacttggcacttaagtgcac 4530 ttttttatga agaaaaagta caatgaactg cttttcctca agcaataattgkttccaact 4590 tgtctgggaa ttgtgtgtct ggtaactgga aggccttcca ctgtggcaaatggaggcttt 4650 tcactgcctg tagagacaat acagtaagca tagttaaggg gtgggtcagaacatgttaag 4710 ataacttact gtatatgtat tcccttgtat tttgttaaag ctggaacatttgatattttt 4770 ccatttattt atgaaaaaat atgaacctat tttcatttgt acaaggtaattgttttttaa 4830 agcaagtcac cttagggtgg ctttaattgt ataagtcaag cacatgtaataaattcaaaa 4890 cctgcagtta acaggatatt agacatcaat cctggtaacc aaatattaaagattctcttt 4950 aaaaaagact gaacatgttt acaggtttga attaggctaa aaggtcttgcagtggctttt 5010 catggccctt caaattggaa tggaactact gtactttgcc atttttctataaatcagtat 5070 ttttttttaa ttttgatata cattgtgtga aaaaagaaaa tggctaataaactgtattaa 5130 atcttaaaca atgtataaag attgtactta gccagttcaa agtgtatatttattcataat 5190 gaattataac agttatattt ttgtgttttc ttgtaaatgt ttcttttcccttaaatacag 5250 ataattcatt tgtattgctt attttattat gagctacaac aaaaggacttcaggaacaag 5310 taatgtatta gtatggttca agattgttga taggaactgt ctcaaaaggatggtggttat 5370 tttaaatata aatagctaat gggggtggta ggcctataaa attaaatgccttgtataaaa 5430 tccaaaatga atgcaaaatt gttttcactt gtattgactt tatgttgtatgattccaatc 5490 tctgttctgt ttggcacttg tatttaattc ttcacctttg taagacatttgtatattgtg 5550 gatgtgttca ttcaagctat ttaatatctg gcactgttaa tacacagtactttattgtac 5610 agactgtttt actgttttaa ttgtagttct gtgtactttt tttggatggggctggcatgt 5670 tttctttgtt tcctggcaat acgacgtggg aatttcaatg cgttttgttgtagatgctaa 5730 cgtgtcagaa tcctttacat tcaacttttc taagaaaagc attttcagtcttgtagtgtg 5790 tgcttacagt aactaatttt gttgaaaatg gtttcaagtt attcaaatttgtacaggact 5850 gtaaagattt gttgacagca aaatgttgaa gaaaaaagct tatagaataaaagctataaa 5910 gtatatatta ggatctgcaa acaatgaaga attatgtaat atattgtacaaatgtaagca 5970 aaggctctga aataaaatgc catagtttgt ga 6002 5 392 DNA Homosapiens 5 ccggagtgag gagctcggtc gccgaagcgg agggagactc ttgagcttcatcttgccgcc 60 gccacggcca ccgcctggac ctttgcccgg agggagctgc agagggtccatcgccgccgt 120 cctctggagg gcagcgcgat tgggggcccg gacctccagt ccgggggggatttttcgtcg 180 tccccctccc cccaaccagg gagcccgagc ggccgccaaa caaaggtaccagtcgccgcc 240 gcgggaggag gaggagccgg agcctctgcc tcagcagccg ctggacccgccgcccttctt 300 ccccatctct cccccgggcc tgctggtttt gggggggaga aggagagaggggactctgga 360 cgtgccaggg tcagatctcg cctccgagga ag 392 6 55 DNA Homosapiens 6 gtgcagctga acctggtgtt ttagaggata ccttggtccc agagtcatca tgaag55 7 111 DNA Homo sapiens 7 gcccttgatg agcctcccta tttgacagtg ggcactgatgtgagtgctaa atacagagga 60 gccttttgtg aagccaagat caagacagca aaaagacttgtcaaagtcaa g 111 8 66 DNA Homo sapiens 8 gtgacattta gacatgattcttcaacagtg gaagttcagg atgaccacat aaagggccca 60 ctaaag 66 9 91 DNA Homosapiens 9 gtaggagcta ttgtggaagt gaagaatctt gatggtgcat atcaggaagctgttatcaat 60 aaactaacag atgcgagttg gtacactgta g 91 10 80 DNA Homosapiens 10 tttttgatga cggagatgag aagacactga gacgatcttc actgtgcctgaaaggagaga 60 ggcattttgc tgaaagtgaa 80 11 92 DNA Homo sapiens 11acattagacc agctcccact caccaaccct gagcattttg gcactccagt cataggaaag 60aaaacaaata gaggaagaag atctaatcat at 92 12 139 DNA Homo sapiens 12accagaggaa gagtcttcat catcctccag tgatgaagat gaggatgata ggaaacagat 60tgatgagcta ctaggcaaag ttgtatgtgt agattacatt agtttggata aaaagaaagc 120actgtggttt cctgcattg 139 13 80 DNA Homo sapiens 13 gtggtttgtc ctgattgtagtgatgagatt gctgtaaaaa aggacaatat tcttgttcga 60 tctttcaaag atggaaaatt 8014 77 DNA Homo sapiens 14 tacttcagtt ccaagaaaag atgtccatga aattactagtgacactgcac caaagcctga 60 tgctgtttta aagcaag 77 15 155 DNA Homo sapiens15 cctttgaaca ggcacttgaa tttcacaaaa gtagaactat tcctgctaac tggaagactg 60aattgaaaga agatagctct agcagtgaag cagaggaaga agaggaggag gaagatgatg 120aaaaagaaaa ggaggataat agcagtgaag aagag 155 16 73 DNA Homo sapiens 16gaagaaatag aaccatttcc agaagaaagg gagaactttc ttcagcaatt gtacaaattt 60atggaagata gag 73 17 95 DNA Homo sapiens 17 gtacacctat taacaaacgacctgtacttg gatatcgaaa tttgaatctc tttaagttat 60 tcagacttgt acacaaacttggaggatttg ataat 95 18 98 DNA Homo sapiens 18 attgaaagtg gagctgtttggaaacaagtc taccaagatc ttggaatccc tgtcttaaat 60 tcagctgcag gatacaatgttaaatgtgct tataaaaa 98 19 244 DNA Homo sapiens 19 atacttatat ggttttgaggagtactgtag atcagccaac attgaatttc agatggcatt 60 gccagagaaa gttgttaacaagcaatgtaa ggagtgtgaa aatgtaaaag aaataaaagt 120 taaggaggaa aatgaaacagagatcaaaga aataaagatg gaggaggaga ggaatataat 180 accaagagaa gaaaagcctattgaggatga aattgaaaga aaagaaaata ttaagccctc 240 tctg 244 20 176 DNA Homosapiens 20 ggaagtaaaa agaatttatt agaatctata cctacacatt ctgatcaggaaaaagaagtt 60 aacattaaaa aaccagaaga caatgaaaat ctggacgaca aagatgatgacacaactagg 120 gtagatgaat ccctcaacat aaaggtagaa gctgaggaag aaaaagcaaaatctgg 176 21 258 DNA Homo sapiens 21 agatgaaacg aataaagaag aagatgaagatgatgaagaa gcagaagagg aggaggagga 60 ggaagaagaa gaagaggatg aagatgatgatgacaacaat gaggaagagg agtttgagtg 120 ctatccacca ggcatgaaag tccaagtgcggtatggacga gggaaaaatc aaaaaatgta 180 tgaagctagt attaaagatt ctgatgttgaaggtggagag gtcctttact tggtgcatta 240 ctgcggatgg aatgtgag 258 22 85 DNAHomo sapiens 22 atacgatgaa tggattaaag cagataaaat agtaagacct gctgataaaaatgtgccaaa 60 gataaaacat cggaagaaaa taaag 85 23 199 DNA Homo sapiens 23aataaattag acaaagaaaa agacaaagat gaaaaatact ctccaaaaaa ctgtaaactt 60cggcgcttgt ccaaaccacc atttcagaca aatccatctc ctgaaatggt atccaaactg 120gatctcactg atgccaaaaa ctctgatact gctcatatta agtccataga aattacttcg 180atccttaatg gacttcaag 199 24 1209 DNA Homo sapiens 24 cttctgaaagttctgctgaa gacagtgagc aggaagatga gagaggtgct caagacatgg 60 ataataatggcaaagaggaa tctaagattg atcatttgac caacaacaga aatgatctta 120 tttcaaaggaggaacagaac agttcatctt tgctagaaga aaacaaagtt catgcagatt 180 tggtaatatccaaaccagtg tcaaaatctc cagaaagatt aaggaaagat atagaagtat 240 tatccgaagatactgattat gaagaagatg aagtcacaaa aaagagaaag gatgtcaaga 300 aggacacaacagataaatct tcaaaaccac aaataaaacg tggtaaaaga aggtattgca 360 atacagaagagtgtctaaaa actggatcac ctggcaaaaa ggaagagaag gccaagaaca 420 aagaatcactttgcatggaa aacagtagca acagctcttc agatgaagat gaagaagaaa 480 caaaagcaaagatgacacca actaagaaat acaatggttt ggaggaaaaa agaaaatctc 540 tacggacaactggtttctat tcaggatttt cagaagtggc agaaaaaagg attaaacttt 600 taaataactctgatgaaaga cttcaaaaca gcagggccaa agatcgaaaa gatgtctggt 660 caagtattcagggacagtgg cctaaaaaaa cgctgaaaga gcttttttca gactctgata 720 ctgaggctgcagcttcccca ccgcatcctg ccccagagga gggggtggca gaggagtcac 780 tgcagactgtggctgaagag gagagttgtt cacccagtgt agaactagaa aaaccacctc 840 cagtcaatgtcgatagtaaa cccattgaag aaaaaacagt agaggtcaat gacagaaaag 900 cagaatttccaagtagtggc agtaattcag tgctaaatac ccctcctact acacctgaat 960 cgccttcatcagtcactgta acagaaggca gccggcagca gtcttctgta acagtatcag 1020 aaccactggctccaaaccaa gaagaggttc gaagtatcaa gagtgaaact gatagcacaa 1080 ttgaggtggatagtgttgct ggggagctcc aagacctcca gtctgaaggg aatagctcgc 1140 cagcaggttttgatgccagt gtgagctcaa gcagtagtaa tcagccagaa ccagaacatc 1200 ctgaaaaag1209 25 114 DNA Homo sapiens 25 cctgtacagg tcagaaaaga gtgaaagatgctcagggagg aggaagttca tcaaaaaagc 60 agaaaagaag ccataaagca acagtggtaaacaacaaaaa gaagggaaaa ggca 114 26 216 DNA Homo sapiens 26 caaatagtagtgatagtgaa gaactttcag ctggtgaaag tataactaag agtcagccag 60 tcaaatcagtttccactgga atgaagtctc atagtaccaa atctcccgca aggacgcagt 120 ctccaggaaaatgtggaaag aatggtgata aggatcctga tctcaaggaa cccagtaatc 180 gattacccaaagtttacaaa tggagttttc agatgt 216 27 147 DNA Homo sapiens 27 cggacctggaaaatatgaca agtgccgaac gcatcacaat tcttcaagaa aaacttcaag 60 aaatcagaaaacattatctg tcattaaaat ctgaagtagc ttccattgat cggaggagaa 120 agcgtttaaagaagaaagag agagaaa 147 28 1750 DNA Homo sapiens 28 gtgctgctac atcctcatcctcctcttcac cttcatccag ttccataaca gctgctgtta 60 tgttaacttt agctgaaccgtcaatgtcca gcgcatcaca aaatggaatg tcagttgagt 120 gcaggtgaca gcaggacttgctaaagcact ttgcacttaa tggctgttga gggccacttt 180 ttttttatac tgcacagtggcacaaaaaaa tatcagacaa gcactatttt atatttaaaa 240 attgtttctt gacaagctgacttggcactt aagtgcactt ttttatgaag aaaaagtaca 300 atgaactgct tttcctcaagcaataattgt ttccaacttg tctgggaatt gtgtgtctgg 360 taactggaag gccttccactgtggcaaatg gaggcttttc actgcctgta gagacaatac 420 agtaagcata gttaaggggtgggtcagaac atgttaagat aacttactgt atatgtattc 480 ccttgtattt tgttaaagctggaacatttg atatttttcc atttatttat gaaaaaatat 540 gaacctattt tcatttgtacaaggtaattg ttttttaaag caagtcacct tagggtggct 600 ttaattgtat aagtcaagcacatgtaataa attcaaaacc tgcagttaac aggatattag 660 acatcaatcc tggtaaccaaatattaaaga ttctctttaa aaaagactga acatgtttac 720 aggtttgaat taggctaaaaggtcttgcag tggcttttca tggcccttca aattggaatg 780 gaactactgt actttgccatttttctataa atcagtattt ttttttaatt ttgatataca 840 ttgtgtgaaa aaagaaaatggctaataaac tgtattaaat cttaaacaat gtataaagat 900 tgtacttagc cagttcaaagtgtatattta ttcataatga attataacag ttatattttt 960 gtgttttctt gtaaatgtttcttttccctt aaatacagat aattcatttg tattgcttat 1020 tttattatga gctacaacaaaaggacttca ggaacaagta atgtattagt atggttcaag 1080 attgttgata ggaactgtctcaaaaggatg gtggttattt taaatataaa tagctaatgg 1140 gggtggtagg cctataaaattaaatgcctt gtataaaatc caaaatgaat gcaaaattgt 1200 tttcacttgt attgactttatgttgtatga ttccaatctc tgttctgttt ggcacttgta 1260 tttaattctt cacctttgtaagacatttgt atattgtgga tgtgttcatt caagctattt 1320 aatatctggc actgttaatacacagtactt tattgtacag actgttttac tgttttaatt 1380 gtagttctgt gtactttttttggatggggc tggcatgttt tctttgtttc ctggcaatac 1440 gacgtgggaa tttcaatgcgttttgttgta gatgctaacg tgtcagaatc ctttacattc 1500 aacttttcta agaaaagcattttcagtctt gtagtgtgtg cttacagtaa ctaattttgt 1560 tgaaaatggt ttcaagttattcaaatttgt acaggactgt aaagatttgt tgacagcaaa 1620 atgttgaaga aaaaagcttatagaataaaa gctataaagt atatattagg atctgcaaac 1680 aatgaagaat tatgtaatatattgtacaaa tgtaagcaaa ggctctgaaa taaaatgcca 1740 tagtttgtga 1750 29 1312PRT Homo sapiens CARBOHYD 294..296 potential 29 Met Lys Ala Leu Asp GluPro Pro Tyr Leu Thr Val Gly Thr Asp Val 1 5 10 15 Ser Ala Lys Tyr ArgGly Ala Phe Cys Glu Ala Lys Ile Lys Thr Ala 20 25 30 Lys Arg Leu Val LysVal Lys Val Thr Phe Arg His Asp Ser Ser Thr 35 40 45 Val Glu Val Gln AspAsp His Ile Lys Gly Pro Leu Lys Val Gly Ala 50 55 60 Ile Val Glu Val LysAsn Leu Asp Gly Ala Tyr Gln Glu Ala Val Ile 65 70 75 80 Asn Lys Leu ThrAsp Ala Ser Trp Tyr Thr Val Val Phe Asp Asp Gly 85 90 95 Asp Glu Lys ThrLeu Arg Arg Ser Ser Leu Cys Leu Lys Gly Glu Arg 100 105 110 His Phe AlaGlu Ser Glu Thr Leu Asp Gln Leu Pro Leu Thr Asn Pro 115 120 125 Glu HisPhe Gly Thr Pro Val Ile Gly Lys Lys Thr Asn Arg Gly Arg 130 135 140 ArgSer Asn His Ile Pro Glu Glu Glu Ser Ser Ser Ser Ser Ser Asp 145 150 155160 Glu Asp Glu Asp Asp Arg Lys Gln Ile Asp Glu Leu Leu Gly Lys Val 165170 175 Val Cys Val Asp Tyr Ile Ser Leu Asp Lys Lys Lys Ala Leu Trp Phe180 185 190 Pro Ala Leu Val Val Cys Pro Asp Cys Ser Asp Glu Ile Ala ValLys 195 200 205 Lys Asp Asn Ile Leu Val Arg Ser Phe Lys Asp Gly Lys PheThr Ser 210 215 220 Val Pro Arg Lys Asp Val His Glu Ile Thr Ser Asp ThrAla Pro Lys 225 230 235 240 Pro Asp Ala Val Leu Lys Gln Ala Phe Glu GlnAla Leu Glu Phe His 245 250 255 Lys Ser Arg Thr Ile Pro Ala Asn Trp LysThr Glu Leu Lys Glu Asp 260 265 270 Ser Ser Ser Ser Glu Ala Glu Glu GluGlu Glu Glu Glu Asp Asp Glu 275 280 285 Lys Glu Lys Glu Xaa Asn Ser SerGlu Glu Glu Glu Glu Ile Glu Pro 290 295 300 Phe Pro Glu Glu Arg Glu AsnPhe Leu Gln Gln Leu Tyr Lys Phe Met 305 310 315 320 Glu Asp Arg Gly ThrPro Ile Asn Lys Arg Pro Val Leu Gly Tyr Arg 325 330 335 Asn Leu Asn LeuPhe Lys Leu Phe Arg Leu Val His Lys Leu Gly Gly 340 345 350 Phe Asp AsnIle Glu Ser Gly Ala Val Trp Lys Gln Val Tyr Gln Asp 355 360 365 Leu GlyIle Pro Val Leu Asn Ser Ala Ala Gly Tyr Asn Val Lys Cys 370 375 380 AlaTyr Lys Lys Tyr Leu Tyr Gly Phe Glu Glu Tyr Cys Arg Ser Ala 385 390 395400 Asn Ile Glu Phe Gln Met Ala Leu Pro Glu Lys Val Val Asn Lys Gln 405410 415 Cys Lys Glu Cys Glu Asn Val Lys Glu Ile Lys Val Lys Glu Glu Asn420 425 430 Glu Thr Glu Ile Lys Glu Ile Lys Met Glu Glu Glu Arg Asn IleIle 435 440 445 Pro Arg Glu Glu Lys Pro Ile Glu Asp Glu Ile Glu Arg LysGlu Asn 450 455 460 Ile Lys Pro Ser Leu Gly Ser Lys Lys Asn Leu Leu GluSer Ile Pro 465 470 475 480 Thr His Ser Asp Gln Glu Lys Glu Val Asn IleLys Lys Pro Glu Asp 485 490 495 Asn Glu Asn Leu Asp Asp Lys Asp Asp AspThr Thr Arg Val Asp Glu 500 505 510 Ser Leu Asn Ile Lys Val Glu Ala GluGlu Glu Lys Ala Lys Ser Gly 515 520 525 Asp Glu Thr Asn Lys Glu Glu AspGlu Asp Asp Glu Glu Ala Glu Glu 530 535 540 Glu Glu Glu Glu Glu Glu GluGlu Glu Asp Glu Asp Asp Asp Asp Asn 545 550 555 560 Asn Glu Glu Glu GluPhe Glu Cys Tyr Pro Pro Gly Met Lys Val Gln 565 570 575 Val Arg Tyr GlyArg Gly Lys Asn Gln Lys Met Tyr Glu Ala Ser Ile 580 585 590 Lys Asp SerAsp Val Glu Gly Gly Glu Val Leu Tyr Leu Val His Tyr 595 600 605 Cys GlyTrp Asn Val Arg Tyr Asp Glu Trp Ile Lys Ala Asp Lys Ile 610 615 620 ValArg Pro Ala Asp Lys Asn Val Pro Lys Ile Lys His Arg Lys Lys 625 630 635640 Ile Lys Asn Lys Leu Asp Lys Glu Lys Asp Lys Asp Glu Lys Tyr Ser 645650 655 Pro Lys Asn Cys Lys Leu Arg Arg Leu Ser Lys Pro Pro Phe Gln Thr660 665 670 Asn Pro Ser Pro Glu Met Val Ser Lys Leu Asp Leu Thr Asp AlaLys 675 680 685 Asn Ser Asp Thr Ala His Ile Lys Ser Ile Glu Ile Thr SerIle Leu 690 695 700 Asn Gly Leu Gln Ala Ser Glu Ser Ser Ala Glu Asp SerGlu Gln Glu 705 710 715 720 Asp Glu Arg Gly Ala Gln Asp Met Asp Asn AsnGly Lys Glu Glu Ser 725 730 735 Lys Ile Asp His Leu Thr Asn Asn Arg AsnAsp Leu Ile Ser Lys Glu 740 745 750 Glu Gln Asn Ser Ser Ser Leu Leu GluGlu Asn Lys Val His Ala Asp 755 760 765 Leu Val Ile Ser Lys Pro Val SerLys Ser Pro Glu Arg Leu Arg Lys 770 775 780 Asp Ile Glu Val Leu Ser GluAsp Thr Asp Tyr Glu Glu Asp Glu Val 785 790 795 800 Thr Lys Lys Arg LysAsp Val Lys Lys Asp Thr Thr Asp Lys Ser Ser 805 810 815 Lys Pro Gln IleLys Arg Gly Lys Arg Arg Tyr Cys Asn Thr Glu Glu 820 825 830 Cys Leu LysThr Gly Ser Pro Gly Lys Lys Glu Glu Lys Ala Lys Asn 835 840 845 Lys GluSer Leu Cys Met Glu Asn Ser Ser Asn Ser Ser Ser Asp Glu 850 855 860 AspGlu Glu Glu Thr Lys Ala Lys Met Thr Pro Thr Lys Lys Tyr Asn 865 870 875880 Gly Leu Glu Glu Lys Arg Lys Ser Leu Arg Thr Thr Gly Phe Tyr Ser 885890 895 Gly Phe Ser Glu Val Ala Glu Lys Arg Ile Lys Leu Leu Asn Asn Ser900 905 910 Asp Glu Arg Leu Gln Asn Ser Arg Ala Lys Asp Arg Lys Asp ValTrp 915 920 925 Ser Ser Ile Gln Gly Gln Trp Pro Lys Lys Thr Leu Lys GluLeu Phe 930 935 940 Ser Asp Ser Asp Thr Glu Ala Ala Ala Ser Pro Pro HisPro Ala Pro 945 950 955 960 Glu Glu Xaa Val Ala Glu Glu Ser Xaa Gln ThrVal Ala Glu Glu Glu 965 970 975 Ser Cys Ser Pro Ser Val Glu Leu Glu LysPro Pro Pro Val Asn Val 980 985 990 Asp Ser Lys Pro Ile Glu Glu Lys ThrVal Glu Val Asn Asp Arg Lys 995 1000 1005 Ala Glu Phe Pro Ser Ser GlySer Asn Ser Val Leu Asn Thr Pro Pro 1010 1015 1020 Thr Thr Pro Glu SerPro Ser Ser Val Thr Val Thr Glu Gly Ser Arg 1025 1030 1035 1040 Gln GlnSer Ser Val Thr Val Ser Glu Pro Leu Ala Pro Asn Gln Glu 1045 1050 1055Glu Val Arg Ser Ile Lys Ser Glu Thr Asp Ser Thr Ile Glu Val Asp 10601065 1070 Ser Val Ala Gly Glu Leu Gln Asp Leu Gln Ser Glu Gly Asn SerSer 1075 1080 1085 Pro Ala Gly Phe Asp Ala Ser Val Ser Ser Ser Ser SerAsn Gln Pro 1090 1095 1100 Glu Pro Glu His Pro Glu Lys Ala Cys Thr GlyGln Lys Arg Val Lys 1105 1110 1115 1120 Asp Ala Gln Gly Gly Gly Ser SerSer Lys Lys Gln Lys Arg Ser His 1125 1130 1135 Lys Ala Thr Val Val AsnAsn Lys Lys Lys Gly Lys Gly Thr Asn Ser 1140 1145 1150 Ser Asp Ser GluGlu Leu Ser Ala Gly Glu Ser Ile Thr Lys Ser Gln 1155 1160 1165 Pro ValLys Ser Val Ser Thr Gly Met Lys Ser His Ser Thr Lys Ser 1170 1175 1180Pro Ala Arg Thr Gln Ser Pro Gly Lys Cys Gly Lys Asn Gly Asp Lys 11851190 1195 1200 Asp Pro Asp Leu Lys Glu Pro Ser Asn Arg Leu Pro Lys ValTyr Lys 1205 1210 1215 Trp Ser Phe Gln Met Ser Asp Leu Glu Asn Met ThrSer Ala Glu Arg 1220 1225 1230 Ile Thr Ile Leu Gln Glu Lys Leu Gln GluIle Arg Lys His Tyr Leu 1235 1240 1245 Ser Leu Lys Ser Glu Val Ala SerIle Asp Arg Arg Arg Lys Arg Leu 1250 1255 1260 Lys Lys Lys Glu Arg GluSer Ala Ala Thr Ser Ser Ser Ser Ser Ser 1265 1270 1275 1280 Pro Ser SerSer Ser Ile Thr Ala Ala Val Met Leu Thr Leu Ala Glu 1285 1290 1295 ProSer Met Ser Ser Ala Ser Gln Asn Gly Met Ser Val Glu Cys Arg 1300 13051310 30 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-124-27330 attcacttct taatacccta gatattatta ctgttactgg wttttat 47 31 47 DNA HomoSapiens allele 1..47 polymorphic fragment 5-127-261 31 ttcagtatacaagagtttaa tttaaaactt tataagttta tgaagaa 47 32 47 DNA Homo Sapiensallele 1..47 polymorphic fragment 5-128-60 32 aaaattgctt gtgtgtgctcccacgtgtgt gtgtgtgcct gtttacc 47 33 48 DNA Homo Sapiens allele 1..48polymorphic fragment 5-129-144 33 cttctcttat aattaaaaaa aatatatagtacttcagttc caagaaaa 48 34 39 DNA Homo Sapiens allele 1..39 polymorphicfragment 5-130-257 34 agatgatgaa aaagaaaagg aggataatag cagtgaaga 39 3547 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-130-276 35gaggataata gcagtgaaga agaagtaagt gaaaacagtt gatacct 47 36 47 DNA HomoSapiens allele 1..47 polymorphic fragment 5-131-395 36 cctagcatagcgcctgtcac gtaacaagta gaaykgagga atttgat 47 37 50 DNA Homo Sapiensallele 1..50 polymorphic fragment 5-133-375 37 ttttctaaag tgtattctatgaatactaga tctatgagaa attctgtgaa 50 38 47 DNA Homo Sapiens allele 1..47polymorphic fragment 5-135-155 38 tattttccat atcctctata aagttccaaaatcaatatat tgtataa 47 39 50 DNA Homo Sapiens allele 1..50 polymorphicfragment 5-135-198 39 ataatattat tctttattat ttgttttttt cttcattaagtgctactttt 50 40 47 DNA Homo Sapiens allele 1..47 polymorphic fragment5-135-357 40 ggttgatacc tcctgttgct aagagataaa ccatggatat aggttga 47 4147 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-136-174 41ccagaagaca atgaaaatct ggacgacaaa gatgatgaca caactag 47 42 47 DNA HomoSapiens allele 1..47 polymorphic fragment 5-140-120 42 ccttatgataaattacgaca tacctttttt cttaacctag aataaat 47 43 49 DNA Homo Sapiensallele 1..49 polymorphic fragment 5-140-348 43 ggacttcaag gtaaacataacaatcgttct gttgcatgca agtatttga 49 44 48 DNA Homo Sapiens allele 1..48polymorphic fragment 5-140-361 44 aaacataaca atcgttctgt tgcatgcaagtatttgattt taatttat 48 45 47 DNA Homo Sapiens allele 1..47 polymorphicfragment 5-143-101 45 aggagggggt ggcagaggag tcaatgcaga ctgtggctgaagaggag 47 46 47 DNA Homo Sapiens allele 1..47 polymorphic fragment5-143-84 46 accgcatcct gccccagagg aggaggtggc agaggagtca ctgcaga 47 47 47DNA Homo Sapiens allele 1..47 polymorphic fragment 5-145-24 47tagtaagttc tgtgacaact tataaatgtc ataaagaaca tgtagtt 47 48 47 DNA HomoSapiens allele 1..47 polymorphic fragment 5-148-352 48 tgcttttcctcaagcaataa ttggttccaa cttgtctggg aattgtg 47 49 47 DNA Homo Sapiensallele 1..47 polymorphic fragment 99-1437-325 49 caagagctga catttactgcatacttaatt tgtgccgaac actgaac 47 50 47 DNA Homo Sapiens allele 1..47polymorphic fragment 99-1442-224 50 attaatctca gtcatatttt ggggtttttttcttctctta taattaa 47 51 47 DNA Homo Sapiens allele 1..47 polymorphicfragment 5-124-273, variant version of SEQ ID30 51 attcacttct taataccctagatgttatta ctgttactgg wttttat 47 52 47 DNA Homo Sapiens allele 1..47polymorphic fragment 5-127-261, variant version of SEQ ID31 52ttcagtatac aagagtttaa tttcaaactt tataagttta tgaagaa 47 53 45 DNA HomoSapiens allele 1..45 polymorphic fragment 5-128-60, variant version ofSEQ ID32 53 54 47 DNA Homo Sapiens allele 1..47 polymorphic fragment5-129-144, variant version of SEQ ID33 54 cttctcttat aattaaaaaaaaatatagta cttcagttcc aagaaaa 47 55 39 DNA Homo Sapiens allele 1..39polymorphic fragment 5-130-257, variant version of SEQ ID34 55agatgatgaa aaagaaaagg agggtaatag cagtgaaga 39 56 47 DNA Homo Sapiensallele 1..47 polymorphic fragment 5-130-276, variant version of SEQ ID3556 gaggataata gcagtgaaga agaggtaagt gaaaacagtt gatacct 47 57 47 DNA HomoSapiens allele 1..47 polymorphic fragment 5-131-395, variant version ofSEQ ID36 57 cctagcatag cgcctgtcac gtatcaagta gaaykgagga atttgat 47 58 49DNA Homo Sapiens allele 1..49 polymorphic fragment 5-133-375, variantversion of SEQ ID37 58 ttttctaaag tgtattctat gatactagat ctatgagaaattctgtgaa 49 59 48 DNA Homo Sapiens allele 1..48 polymorphic fragment5-135-155, variant version of SEQ ID38 59 tattttccat atcctctataaaagttccaa aatcaatata ttgtataa 48 60 46 DNA Homo Sapiens allele 1..46polymorphic fragment 5-135-198, variant version of SEQ ID39 60ataatattat tctttattat ttttttcttc attaagtgct actttt 46 61 47 DNA HomoSapiens allele 1..47 polymorphic fragment 5-135-357, variant version ofSEQ ID40 61 ggttgatacc tcctgttgct aagggataaa ccatggatat aggttga 47 62 47DNA Homo Sapiens allele 1..47 polymorphic fragment 5-136-174, variantversion of SEQ ID41 62 ccagaagaca atgaaaatct ggatgacaaa gatgatgacacaactag 47 63 47 DNA Homo Sapiens allele 1..47 polymorphic fragment5-140-120, variant version of SEQ ID42 63 ccttatgata aattacgacatacttttttt cttaacctag aataaat 47 64 48 DNA Homo Sapiens allele 1..48polymorphic fragment 5-140-348, variant version of SEQ ID43 64ggacttcaag gtaaacataa catcgttctg ttgcatgcaa gtatttga 48 65 46 DNA HomoSapiens allele 1..46 polymorphic fragment 5-140-361, variant version ofSEQ ID44 65 aaacataaca atcgttctgt tgtgcaagta tttgatttta atttat 46 66 47DNA Homo Sapiens allele 1..47 polymorphic fragment 5-143-101, variantversion of SEQ ID45 66 aggagggggt ggcagaggag tcactgcaga ctgtggctgaagaggag 47 67 47 DNA Homo Sapiens allele 1..47 polymorphic fragment5-143-84, variant version of SEQ ID46 67 accgcatcct gccccagaggagggggtggc agaggagtca ctgcaga 47 68 47 DNA Homo Sapiens allele 1..47polymorphic fragment 5-145-24, variant version of SEQ ID47 68 tagtaagttctgtgacaact tatgaatgtc ataaagaaca tgtagtt 47 69 47 DNA Homo Sapiensallele 1..47 polymorphic fragment 5-148-352, variant version of SEQ ID4869 tgcttttcct caagcaataa ttgtttccaa cttgtctggg aattgtg 47 70 47 DNA HomoSapiens allele 1..47 polymorphic fragment 99-1437-325, variant versionof SEQ ID49 70 caagagctga catttactgc atatttaatt tgtgccgaac actgaac 47 7147 DNA Homo Sapiens allele 1..47 polymorphic fragment 99-1442-224,variant version of SEQ ID50 71 attaatctca gtcatatttt gggttttttttcttctctta taattaa 47 72 19 DNA Artificial Sequence primer_bind 1..19upstream amplification primer for SEQ 30, SEQ 51 72 aaaagaaaac aaacccagg19 73 19 DNA Artificial Sequence primer_bind 1..19 upstreamamplification primer for SEQ 31, SEQ 52 73 ataagagttt gggaatacc 19 74 18DNA Artificial Sequence primer_bind 1..18 upstream amplification primerfor SEQ 32, SEQ 53 74 tggaaggatg taggatgc 18 75 18 DNA ArtificialSequence primer_bind 1..18 upstream amplification primer for SEQ 33, SEQ54 75 gctactctgt gtgcaatc 18 76 20 DNA Artificial Sequence primer_bind1..20 upstream amplification primer for SEQ 34, SEQ 55, SEQ 35, SEQ 5676 caaacaataa atgtcagtgg 20 77 19 DNA Artificial Sequence primer_bind1..19 upstream amplification primer for SEQ 36, SEQ 57 77 ggttttgaacagcttagtg 19 78 18 DNA Artificial Sequence primer_bind 1..18 upstreamamplification primer for SEQ 37, SEQ 58 78 tcttttgagt ctaggacc 18 79 18DNA Artificial Sequence primer_bind 1..18 upstream amplification primerfor SEQ 38, SEQ 59, SEQ 39, SEQ 60, SEQ 40, SEQ 61 79 aaagatggaggaggagag 18 80 19 DNA Artificial Sequence primer_bind 1..19 upstreamamplification primer for SEQ 41, SEQ 62 80 tgttgctaag agataaacc 19 81 19DNA Artificial Sequence primer_bind 1..19 upstream amplification primerfor SEQ 42, SEQ 63, SEQ 43, SEQ 64, SEQ 44, SEQ 65 81 gggcttcttatgttctttc 19 82 20 DNA Artificial Sequence primer_bind 1..20 upstreamamplification primer for SEQ 45, SEQ 66, SEQ 46, SEQ 67 82 gcctaaaaaaacgctgaaag 20 83 19 DNA Artificial Sequence primer_bind 1..19 upstreamamplification primer for SEQ 47, SEQ 68 83 tagtaagttc tgtgacaac 19 84 18DNA Artificial Sequence primer_bind 1..18 upstream amplification primerfor SEQ 48, SEQ 69 84 gtttggtctc ccttttcc 18 85 20 DNA ArtificialSequence primer_bind 1..20 upstream amplification primer for SEQ 49, SEQ70 85 caaaagaaac tcacaaagac 20 86 18 DNA Artificial Sequence primer_bind1..18 upstream amplification primer for SEQ 50, SEQ 71 86 tctggtatttgggaacac 18 87 19 DNA Artificial Sequence primer_bind 1..19 downstreamamplification primer for SEQ 30, SEQ 51 87 gtggaaagat aaaatccag 19 88 19DNA Artificial Sequence primer_bind 1..19 downstream amplificationprimer for SEQ 31, SEQ 52 88 caagattttg cctttcctg 19 89 19 DNAArtificial Sequence primer_bind 1..19 downstream amplification primerfor SEQ 32, SEQ 53 89 gaaaaacagt gactctttg 19 90 19 DNA ArtificialSequence primer_bind 1..19 downstream amplification primer for SEQ 33,SEQ 54 90 aataaaggtc acaaggaac 19 91 20 DNA Artificial Sequenceprimer_bind 1..20 downstream amplification primer for SEQ 34, SEQ 55,SEQ 35, SEQ 56 91 atgaggatct caaatacaag 20 92 19 DNA Artificial Sequenceprimer_bind 1..19 downstream amplification primer for SEQ 36, SEQ 57 92gctatcaaat tcctcattc 19 93 20 DNA Artificial Sequence primer_bind 1..20downstream amplification primer for SEQ 37, SEQ 58 93 cacaaaaactttcttcacag 20 94 19 DNA Artificial Sequence primer_bind 1..19 downstreamamplification primer for SEQ 38, SEQ 59, SEQ 39, SEQ 60, SEQ 40, SEQ 6194 ttccctagaa aaagatcag 19 95 19 DNA Artificial Sequence primer_bind1..19 downstream amplification primer for SEQ 41, SEQ 62 95 caaatttctatcagtaggg 19 96 20 DNA Artificial Sequence primer_bind 1..20 downstreamamplification primer for SEQ 42, SEQ 63, SEQ 43, SEQ 64, SEQ 44, SEQ 6596 acagtagttg gtaatgtcac 20 97 18 DNA Artificial Sequence primer_bind1..18 downstream amplification primer for SEQ 45, SEQ 66, SEQ 46, SEQ 6797 agcaacacta tccacctc 18 98 20 DNA Artificial Sequence primer_bind1..20 downstream amplification primer for SEQ 47, SEQ 68 98 gttttttaaagcaagtagcc 20 99 20 DNA Artificial Sequence primer_bind 1..20 downstreamamplification primer for SEQ 48, SEQ 69 99 aaagcctcca tttgccacag 20 10021 DNA Artificial Sequence primer_bind 1..21 downstream amplificationprimer for SEQ 49, SEQ 70 100 attctcattc tctcattttc c 21 101 19 DNAArtificial Sequence primer_bind 1..19 downstream amplification primerfor SEQ 50, SEQ 71 101 aataaaggtc acaaggaac 19 102 19 DNA ArtificialSequence primer_bind 1..19 microsequencing oligo for 5-124-273.mis1 102acttcttaat accctagat 19 103 19 DNA Artificial Sequence primer_bind 1..19microsequencing oligo for 5-127-261.mis1 103 gtatacaaga gtttaattt 19 10419 DNA Artificial Sequence primer_bind 1..19 potential microsequencingoligo for 5-128-60. mis1 104 tgcttgtgtg tgctcccac 19 105 19 DNAArtificial Sequence primer_bind 1..19 potential microsequencing oligofor 5-129-144. mis1 105 ctcttataat taaaaaaaa 19 106 19 DNA ArtificialSequence primer_bind 1..19 microsequencing oligo for 5-130-257.mis1 106gatgaaaaag aaaaggagg 19 107 19 DNA Artificial Sequence primer_bind 1..19potential microsequencing oligo for 5-130-276. mis1 107 ataatagcagtgaagaaga 19 108 19 DNA Artificial Sequence primer_bind 1..19microsequencing oligo for 5-131-395.mis1 108 gcatagcgcc tgtcacgta 19 10919 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for5-133-375.mis1 109 tctaaagtgt attctatga 19 110 19 DNA ArtificialSequence primer_bind 1..19 microsequencing oligo for 5-135-155.mis1 110tttccatatc ctctataaa 19 111 19 DNA Artificial Sequence primer_bind 1..19potential microsequencing oligo for 5-135-198. mis1 111 atattattctttattattt 19 112 19 DNA Artificial Sequence primer_bind 1..19 potentialmicrosequencing oligo for 5-135-357. mis1 112 gatacctcct gttgctaag 19113 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligofor 5-136-174.mis1 113 aagacaatga aaatctgga 19 114 19 DNA ArtificialSequence primer_bind 1..19 microsequencing oligo for 5-140-120.mis1 114atgataaatt acgacatac 19 115 19 DNA Artificial Sequence primer_bind 1..19microsequencing oligo for 5-140-348.mis1 115 cttcaaggta aacataaca 19 11619 DNA Artificial Sequence primer_bind 1..19 potential microsequencingoligo for 5-140-361. mis1 116 cataacaatc gttctgttg 19 117 19 DNAArtificial Sequence primer_bind 1..19 potential microsequencing oligofor 5-143-101. mis1 117 gggggtggca gaggagtca 19 118 19 DNA ArtificialSequence primer_bind 1..19 potential microsequencing oligo for 5-143-84.mis1 118 catcctgccc cagaggagg 19 119 19 DNA Artificial Sequenceprimer_bind 1..19 microsequencing oligo for 5-145-24.mis1 119 aagttctgtgacaacttat 19 120 19 DNA Artificial Sequence primer_bind 1..19 potentialmicrosequencing oligo for 5-148-352. mis1 120 tttcctcaag caataattg 19121 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligofor 99-1437-325.mis1 121 agctgacatt tactgcata 19 122 19 DNA ArtificialSequence primer_bind 1..19 potential microsequencing oligo for99-1442-224.mis1 122 atctcagtca tattttggg 19 123 19 DNA ArtificialSequence primer_bind 1..19 potential microsequencing oligo for5-124-273. mis2 123 aawccagtaa cagtaataa 19 124 19 DNA ArtificialSequence primer_bind 1..19 potential microsequencing oligo for5-127-261. mis2 124 tcataaactt ataaagttt 19 125 15 DNA ArtificialSequence primer_bind 1..15 potential microsequencing oligo for5-130-257. mis2 125 tcttcactgc tatta 15 126 19 DNA Artificial Sequenceprimer_bind 1..19 microsequencing oligo for 5-130-276.mis2 126atcaactgtt ttcacttac 19 127 19 DNA Artificial Sequence primer_bind 1..19potential microsequencing oligo for 5-131-395. mis2 127 aattcctcmrttctacttg 19 128 19 DNA Artificial Sequence primer_bind 1..19microsequencing oligo for 5-135-357.mis2 128 cctatatcca tggtttatc 19 12919 DNA Artificial Sequence primer_bind 1..19 potential microsequencingoligo for 5-136-174. mis2 129 ttgtgtcatc atctttgtc 19 130 19 DNAArtificial Sequence primer_bind 1..19 potential microsequencing oligofor 5-140-120. mis2 130 attctaggtt aagaaaaaa 19 131 19 DNA ArtificialSequence primer_bind 1..19 microsequencing oligo for 5-143-101.mis2 131tcttcagcca cagtctgca 19 132 19 DNA Artificial Sequence primer_bind 1..19microsequencing oligo for 5-143-84.mis2 132 cagtgactcc tctgccacc 19 13319 DNA Artificial Sequence primer_bind 1..19 potential microsequencingoligo for 5-145-24. mis2 133 acatgttctt tatgacatt 19 134 19 DNAArtificial Sequence primer_bind 1..19 potential microsequencing oligofor 5-148-352. mis2 134 attcccagac aagttggaa 19 135 19 DNA ArtificialSequence primer_bind 1..19 potential microsequencing oligo for99-1437-325.mis2 135 agtgttcggc acaaattaa 19 136 19 DNA ArtificialSequence primer_bind 1..19 microsequencing oligo for 99-1442-224.mis2136 ttataagaga agaaaaaaa 19 137 27 DNA Artificial Sequence misc_binding1..27 amplification oligonucleotide hRBBP1.5 137 cccttgatga gcctccctatttgacag 27 138 30 DNA Artificial Sequence misc_binding 1..30amplification oligonucleotide hRBBP1.3 138 cgcattgaaa ttcccacgtcgtattgccag 30 139 18 DNA Artificial Sequence misc_binding 1..18sequencing oligonucleotide PrimerPU 139 tgtaaaacga cggccagt 18 140 18DNA Artificial Sequence misc_binding 1..18 sequencing oligonucleotidePrimerRP 140 caggaaacag ctatgacc 18

What is claimed is:
 1. A purified or isolated polynucleotide comprisinga contiguous span of at least 12 nucleotides of SEQ ID No. 1 or thecomplements thereof, wherein said contiguous span comprises at least 1of the following nucleotide positions of SEQ ID No. 1: 1-481, 666-1465,1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841,81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962,99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, and 161453-162450 2 A purified or isolated polynucleotidecomprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4or the complements thereof, wherein said contiguous span comprises atleast I of the following nucleotide positions of SEQ D No. 4: 1-208,1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and5017-5579.
 3. A purified or isolated nucleic acid comprising anucleotide sequence selected >from the group of SEQ ID Nos. 5-28, asequence complementary thereto or a fragment or a variant thereof.
 4. Apurified or isolated nucleic acid comprising a combination of at leasttwo polynucleotides selected from the group consisting of SEQ ID Nos.5-28, wherein the polynucleotides are arranged within the nucleic acid,from the 5′ end to the 3′end of said nucleic acid, in the same orderthan in SEQ ID No 1
 5. An oligonucleotide of at least 8 nucleotides inlength that hybridizes under stringent hybridization conditions with anucleic acid selected from the group consisting of the nucleotidesequences 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598,72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856,93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084,105180-106682, 106781-107798, 107897-108392, 108552-114335,114418-114491, 114594-132246, 132332-134150, 134350-145565,145842-146332, 146775-150446, 150542-152959, 153176-155590,155738-159701, 160466-161028, and 161453-162450 of SEQ ID No 1 or asequence complementary thereto
 6. An oligonucleotide of at least 8nucleotides in length that hybridizes under stringent hybridizationconditions with a nucleic acid selected from the group consisting of thenucleotide sequences 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333,3871-3882, 4222-4276, and 5017-5579 of SEQ ID No. 4 or a sequencecomplementary thereto
 7. An isolated, purified, or recombinantpolynucleotide consisting essentially of a contiguous span of 8 to 50nucleotides of any one of SEQ ID Nos. 1 and 4 and the complementthereof, wherein said span includes a biallelic marker of RBP-7.
 8. Apolynucleotide according to claim 7, wherein said biallelic marker ofRBP-7 is selected from the group consisting of A1 to A21, and thecomplements thereof
 9. A polynucleotide according to claim 7, whereinsaid contiguous span is 18 to 47 nucleotides in length and saidbiallelic marker is within 4 nucleotides of the center of saidpolynucleotide.
 10. A polynucleotide according to claim 9, wherein saidpolynucleotide consists of said contiguous span and said contiguous spanis 25 nucleotides in length and said biallelic marker is at the centerof said polynucleotide.
 11. A polynucleotide according to claim 9,wherein said polynucleotide consists essentially of a sequence selectedfrom the sequences SEQ ID Nos. 30-71 and the complementary sequencesthereto.
 12. A polynucleotide according to claim 7, wherein the 3′ endof said contiguous span is located at the 3′ end of said polynucleotideand said biallelic marker is present at the 3′ end of saidpolynucleotide.
 13. An isolated, purified, or recombinant polynucleotideconsisting essentially of a contiguous span of 8 to 50 nucleotides ofany one of SEQ ID Nos 1 and 4 and the complement thereof, wherein the 3′end of said contiguous span is located at the 3′ end of saidpolynucleotide, and wherein the 3′ end of said polynucleotide is locatedwithin 20 nucleotides upstream of a biallelic marker of RBP-7 in saidsequence
 14. A polynucleotide according to claim 13, wherein the 3′ endof said polynucleotide is located 1 nucleotide upstream of saidbiallelic marker of RBP-7 in said sequence.
 15. A polynucleotideaccording to claim 14, wherein said polynucleotide consists essentiallyof a sequence selected from the sequences of SEQ ID Nos 102-136. 16 Anisolated, purified, or recombinant polynucleotide consisting essentiallyof a sequence selected from the sequences of SEQ ID Nos. 72-101
 17. Anisolated, purified, or recombinant polynucleotide which encodes apolypeptide comprising a contiguous span of at least 6 amino acids ofSEQ ID No.
 29. 18 An isolated, purified, or recombinant polynucleotidefor use in hybridization assays, sequencing assays, and enzyme-basedmismatch detection assays for determining the identity of the nucleotideat a biallelic marker of RBP-7.
 19. A solid support having apolynucleotide attached thereto, wherein said polynucleotide is selectedfrom the group consisting of a) a polynucleotide comprising a contiguousspan of at least 12 nucleotides of SEQ ID No. 1 or the complementsthereof, wherein said contiguous span comprises at least 1 of thefollowing nucleotide positions of SEQ ID No 1: 1-481, 666-1465,1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841,81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962,99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, and 161453-162450; b) a polynucleotides comprising acontiguous span of at least 12 nucleotides of SEQ ID No 4 or thecomplements thereof, wherein said contiguous span comprises at least 1of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350,1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c)a polynucleotide consisting essentially of a contiguous span of 8 to 50nucleotides of any one of SEQ ID Nos. 1 and 4 and the complementthereof, wherein said span includes a biallelic marker of RBP-7, d) apolynucleotide consisting essentially of a contiguous span of 8 to 50nucleotides of any one of SEQ ID Nos. 1 and 4 and the complementthereof, wherein the 3′ end of said contiguous span is located at the 3′end of said polynucleotide, and wherein the 3′ end of saidpolynucleotide is located within 20 nucleotides upstream of a biallelicmarker of RBP-7 in said sequence; e) a polynucleotide consistingessentially of a sequence selected from the sequences of SEQ ID Nos.72-101, f) a polynucleotide which encodes a polypeptide comprising acontiguous span of at least 6 amino acids of SEQ ID No 29; and g) apolynucleotide for use in hybridization assays, sequencing assays, andenzyme-based mismatch detection assays for determining the identity ofthe nucleotide at a biallelic marker of RBP-7
 20. An array ofpolynucleotides comprising at least one polynucleotide according toclaim 19 21 An array according to claim 20, wherein said array isaddressable.
 22. A polynucleotide having a label thereon, wherein saidpolynucleotide is selected from the group consisting of. a) apolynucleotide comprising a contiguous span of at least 12 nucleotidesof SEQ ID No 1 or the complements thereof, wherein said contiguous spancomprises at least 1 of the following nucleotide positions of SEQ ID No1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543,75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158,97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, and 161453-162450; b) a polynucleotides comprising acontiguous span of at least 12 nucleotides of SEQ ID No 4 or thecomplements thereof, wherein said contiguous span comprises at least 1of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350,1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c)a polynucleotide consisting essentially of a contiguous span of 8 to 50nucleotides of any one of SEQ ID Nos 1 and 4 and the complement thereof,wherein said span includes a biallelic marker of RBP-7, d) apolynucleotide consisting essentially of a contiguous span of 8 to 50nucleotides of any one of SEQ ID Nos. 1 and 4 and the complementthereof, wherein the 3′ end of said contiguous span is located at the 3′end of said polynucleotide, and wherein the 3′ end of saidpolynucleotide is located within 20 nucleotides upstream of a biallelicmarker of RBP-7 in said sequence; e) a polynucleotide consistingessentially of a sequence selected from the sequences of SEQ ID Nos.72-101; f) a polynucleotide which encodes a polypeptide comprising acontiguous span of at least 6 amino acids of SEQ ID No. 29; and g) apolynucleotide for use in hybridization assays, sequencing assays, andenzyme-based mismatch detection assays for determining the identity ofthe nucleotide at a biallelic marker of RBP-7
 23. A recombinant vectorcomprising a polynucleotide selected from the group consisting of: a) apolynucleotide comprising a contiguous span of at least 12 nucleotidesof SEQ ID No. 1 or the complements thereof, wherein said contiguous spancomprises at least 1 of the following nucleotide positions of SEQ ID No.1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543,75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158,97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, and 161453-162450, b) a polynucleotide comprising acontiguous span of at least 12 nucleotides of SEQ ID No 4 or thecomplements thereof, wherein said contiguous span comprises at least 1of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350,1703-1865, 2107-2180, 2843-3333, 3871-3882, 42224276, and 5017-5579; c)a polynucleotide having a sequence selected from the group of SEQ IDNos. 5-28, a sequence complementary thereto or a fragment or a variantthereof; d) a polynucleotide comprising a combination of at least twopolynucleotides selected from the group consisting of SEQ ID Nos. 5-28,wherein the polynucleotides are arranged within the nucleic acid, fromthe 5′ end to the 3 ′end of said nucleic acid, in the same order than inSEQ ID No. 1, and e) a polynucleotide which encodes a polypeptidecomprising a contiguous span of at least 6 amino acids of SEQ ID No. 29.24. A host cell comprising a recombinant vector according to claim 23.25. A non-human host animal or mammal comprising a recombinant vectoraccording to claim
 23. 26. A mammalian host cell comprising a RBP-7 genedisrupted by homologous recombination with a knock out vector,comprising a polynucleotide selected from the group consisting of: a) apolynucleotide comprising a contiguous span of at least 12 nucleotidesof SEQ ID No. 1 or the complements thereof, wherein said contiguous spancomprises at least 1 of the following nucleotide positions of SEQ ID No.1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543,75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158,97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, and 161453-162450, b) a polynucleotide comprising acontiguous span of at least 12 nucleotides of SEQ ID No. 4 or thecomplements thereof, wherein said contiguous span comprises at least 1of the following nucleotide positions of SEQ ID No
 4. 1-208, 1307-1350,1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579, c)a polynucleotide having a sequence selected from the group of SEQ IDNos. 5-28, a sequence complementary thereto or a fragment or a variantthereof; d) a polynucleotide comprising a combination of at least twopolynucleotides selected from the group consisting of SEQ ID Nos 5-28,wherein the polynucleotides are arranged within the nucleic acid, fromthe 5′ end to the 3 ′end of said nucleic acid, in the same order than inSEQ ID No. 1; and e) a polynucleotide which encodes a polypeptidecomprising a contiguous span of at least 6 amino acids of SEQ ID No. 29.27. A non-human host mammal comprising a RBP-7 gene disrupted byhomologous recombination with a knock out vector, comprising apolynucleotide selected from the group consisting of: a) apolynucleotide comprising a contiguous span of at least 12 nucleotidesof SEQ ID No. 1 or the complements thereof, wherein said contiguous spancomprises at least 1 of the following nucleotide positions of SEQ ID No.1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543,75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158,97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682,106781-107798, 107897-108392, 108552-114335, 114418-114491,114594-132246, 132332-134150, 134350-145565, 145842-146332,146775-150446, 150542-152959, 153176-155590, 155738-159701,160466-161028, and 161453-162450; b) a polynucleotide comprising acontiguous span of at least 12 nucleotides of SEQ ID No. 4 or thecomplements thereof, wherein said contiguous span comprises at least 1of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350,1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c)a polynucleotide having a sequence selected from the group of SEQ IDNos. 5-28, a sequence complementary thereto or a fragment or a variantthereof, d) a polynucleotide comprising a combination of at least twopolynucleotides selected from the group consisting of SEQ ID Nos 5-28,wherein the polynucleotides are arranged within the nucleic acid, fromthe 5′ end to the 3′end of said nucleic acid, in the same order than inSEQ ID No 1; and e) a polynucleotide which encodes a polypeptidecomprising a contiguous span of at least 6 amino acids of SEQ ID No. 2928 An isolated, purified, or recombinant polypeptide comprising acontiguous span of at least 6 amino acids of SEQ ID No.
 29. 29 Anisolated or purified antibody composition capable of selectively bindingto an epitope-containing fragment of a polypeptide according to claim 28